Constituents from the bark of Tabebuia impetiginosa

Article abstract of DOI:10.1016/j.phytochem.2004.06.012

The bark of Tabebuia impetiginosa afforded nineteen glycosides, consisting of four iridoid glycosides, two lignan glycosides, two isocoumarin glycosides, three phenylethanoid glycosides and eight phenolic glycosides. Their structures were determined using

Full text of DOI:10.1016/j.phytochem.2004.06.012

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 2003–2011
www.elsevier.com/locate/phytochem
Constituents from the bark of Tabebuia impetiginosa
*
Tsutomu Warashina , Yoshimi Nagatani, Tadataka Noro
Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
Received 23 January 2004; received in revised form 20 April 2004
Available online 7 July 2004
Abstract
The bark of Tabebuia impetiginosa afforded nineteen glycosides, consisting of four iridoid glycosides, two lignan glycosides, two
isocoumarin glycosides, three phenylethanoid glycosides and eight phenolic glycosides. Their structures were determined using both
spectroscopic and chemical methods. Iridoid glycosides, phenylethanoid glycosides and lignan glycosides had ajugol, osmanthuside
H and secoisolariciresinol 4-O-b-D-glucopyranoside as their structural elements, respectively, whereas the aglycone moieties of the
isocoumarin glycosides were considered to be ())-6-hydroxymellein. Phenolic glycosides had 4-methoxyphenol, 2,4-dimethoxy-
phenol, 3,4-dimethoxyphenol, 3,4,5-trimethoxyphenol and vanillyl 4-hydroxybenzoate as each aglycone moiety. Additionally, the
sugar chains of these isocoumarin glycosides and phenolic glycosides were concluded to be b-
pyranoside as well as those of osmanthuside H and above phenylethanoid glycosides.
Ó 2004 Elsevier Ltd. All rights reserved.
D
-apiofuranosyl-(1 ! 6)-b-
D-gluco-
Keywords: Tabebuia impetiginosa; Bignoniaceae; Iridoid; Lignan; Isocoumarin; Phenylethanoid; Phenolic glycoside
1. Introduction
with diethyl ether and partitioned into an ether-soluble
fraction and a water-soluble fraction. The water-soluble
fraction was passed through a porous polymer gel
‘‘Mitsubishi Diaion HP-2000 column and the adsorbed
material was subsequently eluted with MeOH–H2O
(1:1), then MeOH–H2O (7:3) and finally MeOH. The
MeOH–H2O (7:3) eluate from the Diaion HP-20 column
was concentrated and the residue subjected to a silica gel
column and semi-preparative HPLC thereby affording
compounds (1–19). The structural determination of
known compounds 1, 2 and 4 was made based on
comparison of the NMR spectral data with the litera-
ture data (Nakano et al., 1993; Opitz et al., 1999).
Compound 3 was suggested to have the molecular
formula C25H34O13 based on high resolution (HR)-
FABMS [m=z 565.1871 [M + Na]þ]. On comparing the
1H and 13C NMR spectra of 3 with those of known
compounds 1 and 2, signals derived from ajugol (1a)
(Nakano et al., 1993) were also observed. But the proton
signals of the ester moiety resonates at d 7.36 (2H, s) and
d 3.89 (6H, s) and d 3.83 (3H, s), instead of those of the
4-methoxybenzoyl and 3,4-dimethoxybenzoyl groups in
1 and 2. Furthermore, release of 3,4,5-trimethoxyben-
zoic acid with ajugol by alkaline hydrolysis of 3 sug-
Tabebuia impetiginosa is distributed throughout
southeast Latin America and is a national flower of
Brazil (Hashimoto, 1996), and has been used in folk
medicine as a diuretic and an astringent (Hashimoto,
1996). In the course of our research on phytochemi-
cals from Brazilian plants, we have started to study
the constituents present in hydrotropic fraction of the
MeOH extract from the bark of T. impetiginosa. The
present paper describes the isolation and structural
determination of 16 new glycosides along with three
known ones (1 (Nakano et al., 1993), 2 (Nakano
et al., 1993) and 4 (Opitz et al., 1999)).
2. Results and discussion
The MeOH extract from the bark of T. impetiginosa
was suspended in water. The suspension was extracted
* Corresponding author. Tel./fax: +81-54-264-5791.
E-mail address: warashin@sea.u-shizuoka-ken.ac.jp (T. Warash-
ina).
0031-9422/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.06.012

2004
T. Warashina et al. / Phytochemistry 65 (2004) 2003–2011
O
6
3
3"
R1
OH
RO
OCH2
2
1
H
1"
4
5
4"
5"
4
2"
7"
O
A:
B:
5
α
a:
3
6
7
6
5
β
4
1
OH
O
O
O
3
2
HO
9
6"
3"
8
O
3
1
HO
O
O
OMe
OMe
5
2
OH
OH
4
5
H
4
CH2OR2
1"
4"
5"
1
2"
7"
O
10
1
6
5
b:
c:
d:
3
2
6
MeO
MeO
OH
HOCH2
HO
6"
3"
O
6
1
O
4
3
5
4
C:
D:
1"
1
OH
R1 R2
R1 R2
2"
7"
4"
5"
2
3
2
OMe
OMe
HO
5: A
6: A
c
b
d
b
c
a
b
c
13: B
14: E
15: F
16: G
17: G
5a: A
8a: C
11a: D
c
c
c
OMe
OH
MeO
MeO
6"
3"
O
R
1: b
3
7: A
2
1
1"
4
5
2"
7"
4"
5"
2: c
8: C
c
6
3
3: d
9: C
d
H
H
H
O
OMe
OMe
MeO
MeO
4: e
6"
10: C
11: D
12: D
MeO
1a: H
2
OMe
O
4
5
E:
1
6
3
O
O
e:
α
3'
MeO
MeO
2
2'
1'
4'
3'
2'
O
MeO
4
5
F:
1
1'
7'
5'
9'
4'
6
OH
OH
7'
8'
O
O
OH
6'
5'
6'
OH
O
8
8
OH
7
9
1
7
6
O
1
9
G:
2
10
3
4
6
11
3
5
5
OMe
4
6
O
ROCH2
O
1
5
R
4
OH
18: b
19: c
3
2
HO
18a: H
OH
gested that compound 3 consisted of ajugol and the
3,4,5-trimethoxybenzoyl group. Additionally, because
the H-6 signal was observed at d 5.06 (1H, m) as well as 1
and 2, this 3,4,5-trimethoxybenzoyl group was deduced
to be located at the C-6-OH position of ajugol. Ac-
cordingly, the structure of 3 was determined to be 6-O-
(3,4,5-trimethoxybenzoyl)-ajugol.
(d 167.6) and H-5 signals of b- -apiofuranose [d 4.37
D
(1H, d, J ¼ 11:5 Hz) and 4.36 (1H, d, J ¼ 11:5 Hz)]
indicated that the 3,4-dimethoxybenzoyl group was
attached to C-5-OH of b-
ture 5 was elucidated to be 2-(4-hydroxyphenyl)ethyl
1-O-b- -[5-O-(3,4-dimethoxybenzoyl)]-apiofuranosyl-
D-apiofuranose. Thus, struc-
D
(1 ! 6)-b-
D-glucopyranoside. Because of the similarities
1
The molecular formulae of compounds 5, 6 and 7 were
considered to be C28H36O14, C27H34O13 and C29H38O15
on the basis of HR-FABMS, respectively. The 1H NMR
spectra of these three compounds commonly showed the
presence of methylene protons, a glucosyl anomeric
proton, an apiosyl anomeric proton and aromatic pro-
tons. The products from 5–7 by alkaline hydrolysis were
determined to be osmanthuside H (5a) (Sugiyama and
of the H and 13C NMR spectral data of 6 and 7 with
those of 5, osmanthusides I and J (Sugiyama and Ki-
kuchi, 1993) (except for the signals due to the ester
moieties), the structures of 6 and 7 were established as
shown.
Compounds 8, 9 and 10 had the molecular formulae,
C27H34O14, C28H36O15, and C26H32O14, respectively, on
the basis of HR-FABMS analyses. Alkaline hydrolysis
of 8 produced 8a whose molecular formula was deduced
as C19H28O12 from its HR-FABMS, as well as 4-meth-
1
Kikuchi, 1993) by the H and 13C NMR spectral mea-
surements and/or HPLC analysis. At the same time, al-
kaline hydrolysis yielded 3,4-dimethoxybenzoic acid
from 5, 4-methoxybenzoic acid from 6 and 3,4,5-tri-
methoxybenzoic acid from 7. In the heteronuclear mul-
tiple-bond connectivity (HMBC) experiment in 5,
observation of long-range correlations between the car-
bonyl carbon signal of the 3,4-dimethoxybenzoyl group
1
oxybenzoic acid. Comparison of the H and 13C NMR
spectral data of 8a with those of osmanthuside H (5a)
(Sugiyama and Kikuchi, 1993) suggested that the sugar
moiety of 8a was also b-
glucopyranoside. The H NMR spectrum of 8a that the
D
-apiofuranosyl-(1 ! 6)-b-
D-
1
ABX-type aromatic proton signals [d 6.87 (1H, d,

T. Warashina et al. / Phytochemistry 65 (2004) 2003–2011
2005
J ¼ 8:5 Hz), 6.78 (1H, d, J ¼ 2:5 Hz), 6.69 (1H, dd,
tween d 4.67 (H-1 of b-
D
-glucopyranose) and d 7.04; d
J ¼ 8:5, 2.5 Hz)] and two methoxyl proton signals [d
3.82 (3H, s), 3.78 (3H, s)] as the signals due to the
aglycone moiety. In the difference nuclear Overhauser
effect (NOE) experiments in 8a, NOEs were observed as
3.80 (AOMe) and d 6.52; 3.68 (AOMe) and d 6.52, 6.33.
These results suggested that the aglycone of 14 was 2,4-
dimethoxyphenol, and 14 was determined to be 2,4-di-
methoxyphenol 1-O-b-
D
-[5-O-3,4-dimethoxybenzoyl)]-
D-glucopyranoside. Alkaline
follows; d 4.74 (H-1 of b-
D
-glucopyranose) and d 6.78,
apiofuranosyl-(1 ! 6)-b-
6.69; d 3.78 (AOMe) and d 6.87; d 3.82 (AOMe) and d
6.78. These results indicates that the aglycone moiety of
8a was 3,4-dimethoxyphenol, this being confirmed by
acid hydrolysis following alkaline hydrolysis (Section 3).
Accordingly, compound 8a was 3,4-dimethoxyphenyl 1-
hydrolysis of 15 yielded 4-hydroxybenzoic acid with 3,4-
dimethoxybenzoic acid being located at the C-5-OH
position of b-
lowing alkaline hydrolysis afforded vanillyl alcohol
together with -glucose and apiose. In the HMBC
D-apiofuranose, and acid hydrolysis fol-
D
O-b-
compound 8, the attached position of the acyl group was
-apiofuranose, accord-
ing to comparison of the 1H and 13C NMR spectral data
of the apiofuranosyl moiety in 8 with those in 5–7. On
the basis of the above evidence, structure 8 was estab-
D
-apiofuranosyl-(1 ! 6)-b-
D
-glucopyranoside. In
spectrum, long-range correlations were observed be-
tween the carbonyl carbon signal of this 4-hydrox-
ybenzoyl group (d 168.0) and the hydroxy methylene
proton signals of vanillyl alcohol [d 5.13 (2H, s)]. Thus,
the aglycone moiety was deduced to be vanillyl 4-hy-
considered to be C-5-OH of b-
D
droxybenzoate. The attached position of b-
dimethoxybenzoyl)]-apiofuranosyl-(1 ! 6)-b-
pyranosyl unit was confirmed by irradiation of the
anomeric proton signal of b- -glucopyranose in the
D
-[5-O-(3,4-
lished to be 3,4-dimethoxyphenyl 1-O-b-
D
-[5-O-(4-
-glucopyr-
D
-gluco-
methoxybenzoyl)]-apiofuranosyl-(1 ! 6)-b-
D
anoside. Alkaline hydrolysis of 9 and 10, respectively,
produced 3,4-dimethoxybenzoic acid and 4-hydroxy-
benzoic acid together with 8a. Thus, by comparison with
the 1H and 13C NMR spectral data to the apiofuranosyl
moieties in 8–10, the structures of 9 and 10 were deter-
mined as shown.
D
difference NOE experiment. Namely, an NOE was ob-
served for this anomeric proton signal [d 4.83 (1H, d,
J ¼ 8:0 Hz)] and the H-6 signal of vanillyl alcohol [d
7.12 (1H, d, J ¼ 8:0 Hz)]. On above findings, structure
of 15 was established as shown.
Compounds 11 and 12 had the molecular formulae,
C28H36O15 and C29H38O16, respectively, as deduced
from HR-FABMS analyses. The 1H and 13C NMR
spectra of 11 and 12 revealed the presence of one 1,3,4,5-
tetrasubstituted symmetrical aromatic ring, one me-
thoxyl and two equivalent methoxyl groups with the
Compounds 16 and 17 were deduced to have the
molecular formulae, C30H36O16 and C31H38O17 follow-
1
ing the analysis of the HR-FABMS. The H and 13C
NMR spectra revealed signals due to b-
dimethoxybenzoyl)]-apiofuranosyl-(1 ! 6)-b-
pyranosyl unit in 16 and b- -[5-O-(3,4,5-trim-
ethoxybenzoyl)]-apiofuranosyl-(1 ! 6)-b- -glucopyr-
D
-[5-O-(3,4-
D
-gluco-
D
b-
syl unit. Alkaline hydrolysis of 11 afforded 3,4,
5-trimethoxyphenol 1-O-b-
-apiofuranosyl-(1 ! 6)-b-
-glucopyranoside (11a) (Kanchanapoom et al., 2002)
D
-(5-O-acyl)-apiofuranosyl-(1 ! 6)-b-
D
-glucopyrano-
D
anosyl unit in 17, together with the same signals due to
the aglycone moieties in both compounds. Acid hydro-
lysis of 16 afforded the aglycone (16a) which was iden-
tified to be ())-6-hydroxymellein on the basis of
comparison of the 1H NMR, CD spectral data and
optical rotation value (Breinholt et al., 1993; Antus et
al., 1983; Venkatasubbaiah and Chilton, 1991). In the
difference NOE spectrum in 16, irradiation of the ano-
D
D
together with 4-methoxybenzoic acid. Similarly, alkaline
hydrolysis of 12 yielded 11a and 3,4-dimethoxybenzoic
acid. Thus, the structures of 11 and 12 were determined
as shown.
The molecular formulae of compounds 13, 14 and 15
were C27H34O14, C28H36O15 and C35H40O17 on HR-
meric proton signal of b- -glucopyranose [d 4.93 (1H, d,
D
1
FABMS. The analyses of the H and 13C NMR spectra
J ¼ 8:0 Hz)] revealed NOEs corresponding the signals
of H-5 [d 6.39 (1H, d, J ¼ 2:5 Hz)] and H-7 [d 6.51 (1H,
d, J ¼ 2:5 Hz)] of the aglycone. Thus, the above esteri-
fied sugar moiety attached to the C-6 position of ())-6-
hydroxymellein, and the structures of 16 and 17 were
determined to be as shown.
suggested that they had the b-
D
-[5-O-(3,4-dime-
-glucopyrano-
thoxybenzoyl)]-apiofuranosyl-(1 ! 6)-b-
D
syl unit as the sugar and ester moieties. Because acid
hydrolysis following alkaline hydrolysis showed the
aglycone of 13 was 4-methoxyphenol, the structure of 13
was concluded to be 4-methoxyphenyl 1-O-b-
(3,4-dimethoxybenzoyl)]-apiofuranosyl-(1 ! 6)-b-
-glucopyranoside. Compound 14, the AMX-type
aromatic proton signals [d 7.04 (1H, d, J ¼ 9:0 Hz), 6.52
(1H, d, J ¼ 2:5 Hz), 6.33 (1H, dd, J ¼ 9:0, 2.5 Hz)] and
two methoxyl signals [d 3.80 (3H, s), 3.68 (3H, s)] ob-
served in the 1H NMR spectrum indicated that the
aglycone of 14 was a trisubstituted-phenyl group. In the
difference NOE spectra, 14 had showed the NOEs be-
D
-[5-O-
The molecular formulae of compounds 18 and 19
were C34H42O13 and C35H44O14 by HR-FABMS, with
their 1H and 13C NMR spectra of 18 and 19 being
similar to that of glehlinoside A (Yuan et al., 2002).
Additionally, the ester moieties of 18 and 19 were con-
cluded to be 4-methoxybenzoic acid and 3,4-dimeth-
oxybenzoic acid, respectively, according to the
D
1
interpretation of the H, 13C NMR spectral measure-
ments and the results for alkaline hydrolysis; alkaline

2006
T. Warashina et al. / Phytochemistry 65 (2004) 2003–2011
hydrolysis of 18 produced compound 18a, which was
determined to be secoisolariciresinol 4-O-b- -glucopyr-
ilarly, the fractions C (2.1 g) and D (50 mg) afforded
compounds 3 (45 mg), 4 (7 mg), 5 (44 mg), 6 (32 mg), 7
(5 mg), 9 (32 mg), 10 (7 mg), 12 (8 mg) and 1 (25 mg), 2
(14 mg), respectively.
D
anoside by interpretations of the 1H and 13C NMR
spectral data (Yuan et al., 2002) although optical rota-
tion data for 18a differs from the literature value (Yuan
et al., 2002). Thus, it was not possible to assign absolute
configuration of the aglycone moiety of 18 as either 8R,
80R-secoisolariciresinol or 8S, 80S-secoisolariciresinol.
Acid hydrolysis of 18a and 18 could not be carried out
due to insufficient materials. Thus, the deduced struc-
tures of 18 and 19 are as shown.
22
Compound 3: Amorphous powder. ½aꢀD – 130°
MeOH
max
(MeOH; c 2.65). UV k
nm (log e): 213 (4.53), 265
(4.03), 286 (sh). FABMS m=z: 565 [M + Na]þ, HR-
FABMS m=z: 565.1871 (Calcd. for C25H34O13Na:
565.1897). 13C NMR (MeOH-d4 at 35 °C): d 167.4 (C-
100), 154.4 ꢁ 2 (C-400, -600), 143.8 (C-500), 141.3 (C-3), 126.9
(C-200), 108.3 ꢁ 2 (C-300, -700), 104.5 (C-4), 99.4 (CGlc-1),
93.5 (C-1), 81.2 (C-6), 79.2 (C-8), 78.2, 78.0 (CGlc-3, -5),
74.8 (CGlc-2), 71.8 (CGlc-4), 62.9 (CGlc-6), 61.2 (C-500–
OMe), 56.8 ꢁ 2 (C-400, -600–OMe), 51.9 (C-9), 47.7 (C-7),
39.6 (C-5), 26.2 (C-10). 1H NMR (MeOH-d4 at 35 °C): d
7.36 (2H, s, H-300, -700), 6.24 (1H, dd, J ¼ 6:5, 2.5 Hz, H-
3), 5.06 (1H, m, H-6), 5.51 (1H, d, J ¼ 2:5 Hz, H-1), 5.00
(1H, dd, J ¼ 6:5, 2.5 Hz, H-4), 4.68 (1H, d, J ¼ 8:0 Hz,
3. Experimental
Optical rotation and circular dichroism measure-
ments were obtained using a JASCO-DIP1000 digital
polarimeter and a JASCO J-20A automatic recording
spectropolarimeter, respectively. FABMS spectra were
collected on a JEOL JMS-700 spectrometer in m-nitro-
H
Glc-1), 3.90 (overlapping, HGlc-6), 3.89 (6H, s, C-400, -
600–OMe), 3.83 (3H, s, C-500–OMe), 3.67 (1H, dd,
1
benzyl alcohol, whereas both H and 13C NMR spectra
J ¼ 12:0, 5.5 Hz, HGlc-6), 3.38 (1H, t, J ¼ 8:0 Hz, HGlc
-
were recorded in MeOH-d4 solution on a JEOL JNM
A-400 (400 and 100.40 MHz, respectively), spectrome-
ter. Chemical shifts are given in the d (ppm) with tet-
ramethylsilane (TMS) as an internal standard, UV
spectra measured a Beckman UV640 spectrometer,
whereas CG utilized a Hitachi G-3000 gas chromato-
graph and HPLC analyses employed a JASCO 800
system instrument.
3), 3.32 (1H, m, HGlc-5), 3.27 (1H, t, J ¼ 8:0 Hz, HGlc-4),
3.20 (1H, t, J ¼ 8:0 Hz, HGlc-2), 3.02 (1H, m, H-5), 2.63
(1H, dd, J ¼ 9:5, 2.5 Hz, H-9), 2.29 (1H, dd, J ¼ 14:5,
6.0 Hz, H-7), 2.07 (1H, dd, J ¼ 14:5, 3.5 Hz, H-7), 1.42
(3H, s, H-10).
22
Compound 5: Amorphous powder. ½aꢀD –51.6°
max
MeOH
(MeOH; c 2.15). UV k
nm (log e): 201 (4.46), 219
(4.39), 261 (4.03), 286 (3.76). FABMS m=z: 619
[M + Na]þ, HR-FABMS m=z: 619.2007 (Calcd. for
C28H36O14Na: 619.2002). For 13C and 1H NMR spectra,
see Tables1 and2. 1H NMR spectrum ofaglycone moiety:
d 7.00 (2H, d, J ¼ 8:0 Hz, H-2, -6), 6.67 (2H, d, J ¼ 8:0
Hz, H-3, -5), 3.94 (1H, ddd, J ¼ 9:5, 8.0, 6.5 Hz, H-a), 3.67
(1H, ddd, J ¼ 9:5, 8.0, 6.5 Hz, H-a), 2.77 (2H, m, H-b).
3.1. Plant material
The bark of Tabebuia impetiginosa was purchased
in San Paulo-city, Brazil, in 2000, and identified by
Y. Nagatani.
22
Compound 6: Amorphous powder. ½aꢀD –55.4°
MeOH
max
3.2. Extraction and isolation
(MeOH; c 1.93). UV k
nm (log e): 202 (4.88), 219
(sh), 256 (4.48). FABMS m=z: 589 [M + Na]þ, HR-
FABMS m=z: 589.1893 (Calcd. for C27H34O13Na:
The bark of Tabebuia impetiginosa (935 g) was ex-
tracted three times with MeOH under reflux. The extract
was then concentrated under reduced pressure with the
resulting residue suspended in H2O. Following its ex-
traction with Et2O, the H2O layer was passed through a
porous polymer gel ‘‘Mitsubishi Diaion HP-20’’ column
with adsorbed material being eluted with MeOH–H2O
(1:1), MeOH–H2O (7:3) and MeOH, respectively. The
MeOH–H2O (7:3) fraction from the Diaion HP-20 col-
umn was then evaporated to dryness, with the residue
(23.0 g) subject to silica gel CC with a CHCl3–MeOH–
EtOAc–H2O (50:10:38:1) eluant to get five fractions (A
(1.9 g), B (1.6 g), C (6.0 g), D (4.0 g) and E (8.0 g). Using
semi-preparative HPLC (Develosil-ODS-15/30, AC8
and YMC-ODS: 25–30% MeCN in water and 40–50%
MeOH in water), the fraction B (1.6 g) afforded com-
pounds 8 (40 mg), 11 (65 mg), 13 (4 mg), 14 (4 mg), 15 (9
mg), 16 (32 mg), 17 (4 mg), 18 (6 mg), 19 (4 mg). Sim-
1
589.1897). For 13C and H NMR spectra, see Tables 1
1
and 2. The H NMR spectrum of the aglycone moiety
was in good agreement with that of 5.
Compound 7: Amorphous powder. ½aꢀ2D2 – 54° (MeOH;
MeOH
max
c 0.51). UV k
nm (log e): 214 (4.56), 267 (4.05), 291
(sh). FABMS m=z: 649 [M + Na]þ, HR-FABMS m=z:
649.2114 (Calcd. for C29H38O15Na: 649.2108). For 13C
and 1H NMR spectra, see Tables 1 and 2. The 1H NMR
spectrum of the aglycone moiety was in good agreement
with that of 5.
22
Compound 8: Amorphous powder. ½aꢀD –76.0°
max
MeOH
(MeOH; c 1.79). UV k
nm (log e): 202 (4.89), 230
(3.96), 256 (4.20). FABMS m=z: 605 [M + Na]þ, HR-
FABMS m=z: 605.1836 (Calcd. for C27H34O14Na:
1
605.1846). For 13C and H NMR spectra, see Tables 1
and 2. 1H NMR spectrum of the aglycone moiety: d 6.77
(1H, d, J ¼ 8:5 Hz, H-5), 6.74 (1H, d, J ¼ 2:5 Hz, H-2),

Table 1
13C NMR spectral data for compounds 5–17
No.
5
6
7
8
9
10
11
12
13
14
15
67.2
16
17
C-a
-b
-1
72.1
36.4
72.1
36.4
72.1
36.4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
155.9
96.7
–
155.9
96.7
–
–
171.2
–
171.2
130.8
130.9
130.8
130.9
130.8
130.9
153.8
104.5
153.8
104.5
153.8
104.5
153.2
119.3
142.0
152.1
147.9
150.9
113.9
132.6
122.3
118.1
–
-2
–
–
-3
116.1
156.7
116.1
156.7
116.1
156.8
151.1
146.2
151.1
146.2
151.1
146.2
154.8
134.8
154.8
134.9
115.5
156.6
101.6
157.6
77.2a
35.4
108.6
165.1b
103.4
164.9b
103.8
143.1
20.8
–
77.2a
35.4
108.6
165.2b
103.1
164.9b
103.7
143.0
20.8
–
-4
-5
116.1
130.9
116.1
130.9
116.1
130.9
114.0
109.4
114.0
109.3
114.1
109.4
154.8
96.7
154.8
96.7
115.5
119.3
105.4
120.1
-6
-7
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
-8
–
–
–
–
–
-9
–
–
–
–
–
–
–
–
-10
-11
AOMe
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
56.0
56.7a
–
56.5
57.1
–
56.5a
56.6
57.2
–
56.7 ꢁ 2
56.7 ꢁ 2
56.0
–
56.8
–
57.1
–
61.2
–
61.2
–
–
–
-10
-20
-30
-40
-50
-60
-70
–
–
–
–
–
–
–
–
168.0
122.3
132.8
116.2
163.6
116.2
132.8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Sugar moieties
Glc-1
-2
104.4
75.1a
78.1
71.8
76.8
68.6
110.7
78.6
79.0
75.0a
67.8
104.4
75.1a
78.1
71.7
76.8
68.6
110.7
78.5
79.0
75.0a
67.6
104.4
75.1a
78.1
71.8
76.9
68.6
110.8
78.6
79.1
75.0a
68.3
103.5
75.0a
78.0
71.7
76.9
68.5
110.7
78.6
78.9
74.9a
67.6
103.5
75.0b
78.0
71.7
76.9
68.6
110.7
78.7
79.0
74.9b
67.7
103.4
75.1a
78.0
71.7
76.9
68.6
110.7
78.6
79.0
75.0a
67.5
103.3
75.0a
77.9
71.5
76.9
68.3
110.5
78.6
79.0
74.9a
67.6
103.3
75.0a
78.0
71.5
76.9
68.3
110.5
78.7
79.0
74.9a
67.8
103.5
75.0a
78.1
71.8
76.9
68.7
110.8
78.7
79.0
75.0a
67.7
104.1
75.0
77.9
71.8
77.0
68.7
110.8
78.7
79.0
75.0
67.6
102.8
75.1a
77.9
71.8
77.0
68.8
110.9
78.6
79.0
74.9a
67.6
101.3
74.7c
77.9
101.2
74.7c
77.9
71.8
77.1a
69.5
111.4
78.9
78.9
75.1c
67.7
-3
-4
71.7
-5
77.1a
69.0
-6
Api-1
-2
111.0
78.8d
78.9d
75.1c
67.6
-3
-4
-5
Ester moieties
C-100
-200
167.6
123.5
113.7
150.2
155.0
112.1
125.1
56.5
167.6
123.3
132.8
114.8
165.3
114.8
132.8
56.0
167.4
126.4
108.4
154.5
143.9
154.5
108.4
56.9 ꢁ 2
61.2
167.6
123.3
132.8
114.9
165.3
114.9
132.8
56.0
167.6
123.5
113.8
150.2
155.0
112.1
125.1
56.5
167.9
122.1
133.0
116.2
163.7
116.2
133.0
–
167.6
123.3
132.8
114.9
165.3
114.9
132.8
56.0
167.7
123.5
113.8
150.3
155.0
112.1
125.1
56.5
167.7
123.5
113.8
150.3
155.1
112.1
125.2
56.5
167.6
123.5
113.8
150.2
155.0
112.1
125.1
56.5a
56.6a
167.6
123.5
113.8
150.2
155.0
112.1
125.1
56.5
167.6
123.5
113.7
150.1
154.9
112.0
125.1
56.5 ꢁ 2
–
167.3
126.3
108.3
154.3
143.7
154.3
108.3
56.8 ꢁ 2
61.2
-300
-400
-500
-600
-700
AOMe
56.6
–
–
56.6a
–
–
56.6
56.6
56.6
Measured in MeOH-d4 solution at 35 °C.
Interchangeable in each column.
a–d

Table 2
1H NMR spectral data of the sugar and ester moieties of compounds 5–19
No.
5
6
7
8
9
10
11
Sugar moieties
Glc-1
-2
4.26 (d, 8.0)
3.19 (t, 8.0)
4.26 (d, 8.0)
3.19 (t, 8.0)
3.34 (t, 8.0)
3.28 (t, 8.0)
3.40 (m)
4.25 (d, 8.0)
3.18 (t, 8.0)
3.34a
4.74a
4.72 (d, 8.0)
3.42a
4.74a
4.79 (d, 8.0)
3.44a
3.44a
3.43a
3.43a
-3
3.34 (t, 8.0)
3.43a
3.42a
3.43a
3.33a
-4
3.28 (t, 8.0)
3.26 (t, 8.5)
3.40 (m)
3.34 (t, 8.5)
3.54 (m)
3.33a
3.35 (t, 8.5)
3.56 (m)
-5
3.40 (m)
3.54 (m)
3.54 (m)
-6
4.01 (dd, 11.5, 2.0)
3.63 (dd, 11.5, 6.0)
5.05 (d, 2.0)
4.01 (d, 2.0)
4.09 (d, 10.0)
3.88 (d, 10.0)
4.37 (d, 11.5)
4.36 (d, 11.5)
4.01 (dd, 11.0, 2.0)
3.63 (dd, 11.0, 6.0)
5.05 (d, 2.0)
4.00 (d, 2.0)
4.09 (d, 10.0)
3.87 (d, 10.0)
4.37 (d, 11.5)
4.34 (d, 11.5)
4.00 (dd, 10.5, 2.0)
3.63 (dd, 10.5, 6.0)
5.05 (d, 2.0)
4.00 (d, 2.0)
4.08 (d, 10.0)
3.88 (d, 10.0)
4.40 (d, 11.5)
4.38 (d, 11.5)
4.05 (dd, 11.5, 2.0)
3.63 (dd, 11.5, 6.5)
5.02 (d, 2.0)
3.99 (d, 2.0)
4.08 (d, 10.0)
3.86 (d, 10.0)
4.38 (d, 12.0)
4.34 (d, 12.0)
4.05 (dd, 11.0, 2.0)
3.62 (dd, 11.0, 6.5)
5.02 (d, 2.0)
4.00 (d, 2.0)
4.09 (d, 10.0)
3.86 (d, 10.0)
4.39 (d, 12.0)
4.35 (d, 12.0)
4.05 (dd, 10.5, 2.0)
3.62 (dd, 10.5, 6.5)
5.02 (d, 2.0)
3.98 (d, 2.0)
4.07 (d, 10.0)
3.86 (d, 10.0)
4.45 (d, 11.5)
4.43 (d, 11.5)
4.06 (dd, 11.5, 2.0)
3.63 (dd, 11.5, 6.5)
5.01 (d, 2.0)
3.96 (d, 2.0)
4.08 (d, 10.0)
3.86 (d, 10.0)
4.36 (d, 11.5)
4.31 (d, 11.5)
Api-1
-2
-4
-5
Ester 0m0 oieties
-3
-400
7.56 (d, 2.0)
–
7.98 (brd, 8.5)
6.97 (brd, 8.5)
6.97 (brd, 8.5)
7.98 (brd, 8.5)
3.85 (s)
7.35 (s )
–
7.99 (d, 8.5)
6.97 (d, 8.5)
6.97 (d, 8.5)
7.99 (d, 8.5)
3.84 (s)
7.56 (d, 2.0)
–
7.91 (brd, 8.5)
6.82 (brd, 8.5)
6.82 (brd, 8.5)
7.91 (brd, 8.5)
7.95 (d, 8.5)
6.97 (d, 8.5)
6.97 (d, 8.5)
7.95 (d, 8.5)
3.85 (s)
-600
6.99 (d, 8.0)
7.69 (dd, 8.0, 2.0)
3.88 (s)
–
6.99 (d, 8.0)
7.69 (dd, 8.0, 2.0)
3.88 (s)
-700
7.35 (s)
3.87 (s)x2
3.82 (s)
AOMes
3.85 (s)
–
–
3.84 (s)
–
No.
12
13
14
15
16
17
18
19
Sugar moieties
Glc-1
-2
4.77 (d, 8.0)
4.69 (d, 8.0)
3.41a
4.67 (d, 8.5)
3.41 (t, 8.5)
3.44 (t, 8.5)
3.32a
4.83 (d, 8.0)
3.50 (t, 8.0)
3.44 (t, 8.0)
3.32a
4.93 (d, 8.0)
3.46a
4.93 (d, 8.0)
3.45a
4.85 (d, 8.0)
4.84 (d, 8.0)
3.43a
3.52a
3.52a
-3
3.43a
3.41a
3.46a
3.45a
3.52a
3.52a
-4
3.35 (t, 8.5)
3.55 (m)
3.32a
3.33a
3.27a
3.44 (t, 8.5)
3.44 (t, 8.5)
-5
3.52 (m)
3.48 (m)
3.56 (m)
3.67 (m)
3.71 (m)
3.75 (m)
4.64 (dd, 12.0, 2.5)
4.41 (dd, 12.0, 7.5)
3.75 (m)
4.65 (dd, 12.0, 2.5)
4.44 (dd, 12.0, 7.5)
-6
4.06 (dd, 11.0, 2.0)
3.63 (dd, 11.0, 6.0)
5.01 (d, 2.0)
3.95 (d, 2.0)
4.08 (d, 10.0)
3.85 (d, 10.0)
4.37 (d, 11.5)
4.32 (d, 11.5)
4.04 (dd, 11.0, 2.0)
3.62 (dd, 11.0, 6.5)
5.02 (d, 2.0)
4.02 (d, 2.0)
4.09 (d, 10.0)
3.87 (d, 10.0)
4.40 (d, 11.0)
4.35 (d, 11.0)
4.01 (dd, 11.0, 2.0)
3.62 (dd, 11.0, 6.5)
5.01 (d, 2.0)
4.00 (d, 2.0)
4.06 (d, 10.0)
3.85 (d, 10.5)
4.37 (d, 11.5)
4.33 (d, 11.5)
4.04 (dd, 11.0, 2.0)
3.61 (dd, 11.0, 6.5)
5.00 (d, 2.0)
4.00 (d, 2.0)
4.07 (d, 10.0)
3.86 (d, 10.0)
4.38 (d, 11.5)
4.34 (d, 11.5)
4.05 (brd, 10.5)
3.63 (dd, 10.5, 7.5)
5.01 (d, 2.0)
4.04 (d, 2.0)
4.17 (d, 10.0)
3.88 (d, 10.0)
4.37 (d, 12.0)
4.34 (d, 12.0)
4.05 (dd, 11.5, 2.0)
3.64 (dd, 11.5, 7.5)
5.02 (d, 2.0)
4.09 (d, 2.0)
4.21 (d, 10.0)
3.88 (d, 10.0)
4.37 (d, 11.5)
4.35 (d, 11.5)
Api-1
-2
–
–
–
–
–
–
–
–
–
–
–
–
-4
-5
Ester 0m0 oieties
-3
-400
7.57 (d, 2.0)
–
7.57 (d, 2.0)
–
7.56 (d, 2.0)
–
7.54 (d, 2.0)
–
7.52 (d, 2.0)
–
7.30 (s)
–
7.96 (d, 8.0)
6.97 (d, 8.0)
6.97 (d, 8.0)
7.96 (d, 8.0)
3.86 (s)
7.53 (d, 2.0)
–
-600
7.00 (d, 8.5)
7.68 (dd, 8.5, 2.0)
3.89 (s)
7.01 (d, 8.0)
7.71 (dd, 8.0, 2.0)
3.88 (s)
7.01 (d, 8.0)
7.69 (dd, 8.0, 2.0)
3.88 (s)
6.96 (d, 8.5)
7.68 (dd, 8.5, 2.0)
3.85 (s)
6.97 (d, 8.5)
7.66 (dd, 8.5, 2.0)
3.88 (s)
–
7.00 (d, 8.0)
7.68 (dd, 8.0 2.0)
3.89 (s)
-700
7.30 (s )
3.84 (s)x2
3.81 (s)
AOMes
3.85 (s)
3.84 (s)
3.86 (s)
3.80 (s)
3.82 (s)
–
3.82 (s)
Measured in MeOH-d4 solution at 35 °C.
a Overlapping with other signals.

T. Warashina et al. / Phytochemistry 65 (2004) 2003–2011
2009
6.64 (1H, dd, J ¼ 8:5, 2.5 Hz, H-6), 3.77 (3H, s, –OMe),
3.71 (3H, s, –OMe).
H-6), 7.03 (1H, d, J ¼ 2.0 Hz, H-3), 6.88 (1H, dd,
J ¼ 8:0, 2.0 Hz, H-5), 6.80 (2H, br d, J ¼ 8:5 Hz, H-40, -
60), 5.13 (2H, s, H-a), 3.84 (3H, s, AOMe).
22
Compound 9: Amorphous powder. ½aꢀD –72.0°
max
22
MeOH
(MeOH; c 1.89). UV k
nm (log e): 201 (4.88), 219
Compound 16: Amorphous powder. ½aꢀD –94.8°
max
MeOH
(4.41), 261 (4.07), 286 (3.88). FABMS m=z: 635
[M + Na]þ, HR-FABMS m=z: 635.1951 (Calcd. for
C28H36O15Na: 635.1952). For 13C and 1H NMR
spectra, see Tables 1 and 2. The 1H NMR spectrum
of the aglycone moiety was in good agreement with
that of 8.
(MeOH; c 1.59). UV k
nm (log e): 201 (4.87), 215
(4.57), 261 (4.29), 296 (3.91). FABMS m=z: 653
[M + H]þ, 675 [M + Na]þ, HR-FABMS m=z: 653.2086,
675.1887
(Calcd.
for
C30H37O16:
653.2082,
C30H36O16Na: 675.1887). For 13C and 1H NMR spectra,
1
see Tables 1 and 2. H NMR spectrum of the aglycone
22
Compound 10: Amorphous powder. ½aꢀD –75°
moiety: d 6.51 (1H, d, J ¼ 2:5 Hz, H-7), 6.39 (1H, d,
J ¼ 2:5 Hz, H-5), 4.59 (1H, m, H-3), 2.86 (1H, dd,
J ¼ 16:0, 4.0 Hz, H-4), 2.79 (1H, dd, J ¼ 16:0, 10.5 Hz,
H-4), 1.42 (3H, d, J ¼ 6:5 Hz, H-11).
MeOH
max
(MeOH; c 0.66). UV k
nm (log e): 202 (4.88), 258
(4.15). FABMS m=z: 591 [M + Na]þ, HR-FABMS m=z:
591.1661 (Calcd. for C26H32O14Na: 591.1689). For 13C
and 1H NMR spectra, see Tables 1 and 2. The 1H NMR
spectrum of the aglycone moiety was in good agreement
with that of 8.
24
Compound 17: Amorphous powder. ½aꢀD –95°
MeOH
max
(MeOH; c 0.40). UV k
nm (log e): 213 (4.77), 262
(4.40), 295 (sh). FABMS m=z: 705 [M + Na]þ, HR-
FABMS m=z: 705.1976 (Calcd. for C31H38O17Na:
22
Compound 11: Amorphous powder. ½aꢀD –79.1°
max
MeOH
1
(MeOH; c 2.05). UV k
nm (log e): 203 (4.97), 256
705.2007). For 13C and H NMR spectra, see Tables 1
(4.20). FABMS m=z: 635 [M + Na]þ, HR-FABMS m=z:
and 2. The H NMR spectrum of the aglycone moiety
was in good agreement with that of 16.
1
635.1929 (Calcd. for C28H36O15Na: 635.1952). For 13C
22
1
1
and H NMR spectra, see Tables 1 and 2. H NMR
spectrum of the aglycone moiety: d 6.43 (2H, s, H-2, -6),
3.77 (6H, s, –OMes), 3.68 (3H, s, –OMe).
Compound 18: Amorphous powder. ½aꢀD –35°
MeOH
(MeOH; c 0.63). UV k
nm (log e): 203 (5.01), 226
max
(4.16), 257 (4.18). FABMS m=z: 659 [M+H]þ, 681
[M + Na]þ, HR-FABMS m=z: 659.2684, 681.2493
(Calcd. for C34H43O13: 659.2704, C34H42O13Na:
681.2523). 13C NMR (MeOH-d4 at 35 °C): d 167.7 (C-
100), 165.3 (C-500), 150.5 (C-3), 148.8 (C-30), 145.9 (C-4),
145.5 (C-40), 137.4 (C-1), 133.8 (C-10), 132.8 ꢁ 2 (C-300, -
700), 123.5 (C-200), 122.8, 122.7 (C-6, -60), 117.7 (C-5),
115.8 (C-50), 114.9 ꢁ 2 (C-400, -600), 114.5 (C-2), 113.4 (C-
20), 102.8 (CGlc-1), 77.9 (CGlc-3), 75.6 (CGlc-5), 74.9
(CGlc-2), 72.1 (CGlc-4), 65.1 (CGlc-6), 62.2 ꢁ 2 (C-9, -90),
56.6, 56.4 (C-3–OMe, -30–OMe), 56.1 (C-50–OMe),
44.1 ꢁ 2 (C-8, -80), 36.3, 36.2 (C-7, -70). 1H NMR: shown
22
Compound 12: Amorphous powder. ½aꢀD –78°
MeOH
(MeOH; c 0.76). UV k
nm (log e): 203 (5.00), 213
max
(sh), 261 (4.08), 291 (3.76). FABMS m=z: 643 [M + H]þ,
665 [M + Na]þ, HR-FABMS m=z: 643.2223, 665.2051
(Calcd. for C29H39O16: 643.2238, C29H38O16Na:
1
665.2057). For 13C and H NMR spectra, see Tables 1
1
and 2. The H NMR spectrum of the aglycone moiety
was in good agreement with that of 11.
25
Compound 13: Amorphous powder. ½aꢀD –83°
max
MeOH
(MeOH; c 0.39). UV k
nm (log e): 203 (4.48), 220
(4.49), 261 (4.19), 287 (3.92). FABMS m=z: 605
[M + Na]þ, HR-FABMS m=z: 605.1817 (Calcd. for
C27H34O14Na: 605.1846). For 13C and 1H NMR spectra,
1
in Table 2. The H NMR spectral data of the aglycone
moiety (MeOH-d4 at 35 °C): d 6.90 (1H, d, J ¼ 8:0 Hz,
H-5), 6.65 (1H, d, J ¼ 2:0 Hz, H-2), 6.65 (1H, d, J ¼ 8:0
Hz, H-50), 6.52 (1H, d, J ¼ 2:0 Hz, H-20), 6.52 (1H, dd,
J ¼ 8:0, 2.0 Hz, H-60), 6.39 (1H, dd, J ¼ 8:0, 2.0 Hz, H-
6), 3.74 (3H, s, C-3–OMe), 3.69 (3H, s, C-30–OMe), 3.58
(4H, d, J ¼ 5:0Hz, H-9, -90), 2.66 (1H, dd, J ¼ 13:5, 6.5
Hz, H-7), 2.64 (1H, dd, J ¼ 13.5, 6.5 Hz, H-70), 2.54
(1H, dd, J ¼ 13:5, 8.0 Hz, H-7), 2.52 (1H, dd, J ¼ 13:5,
8.0 Hz, H-70), 1.85 (2H, m, H-8, -80).
1
see Tables 1 and 2. H NMR spectrum of the aglycone
moiety: d 6.99 (2H, d, J ¼ 9:0 Hz, H-2, -6), 6.75 (2H, d,
23
J ¼ 9:0 Hz, H-3, -5), 3.67 (3H, s, AOMe).
MeOH
Compound 14: Amorphous powder. ½aꢀD –74°
max
(MeOH; c 0.43). UV k
nm (log e): 202 (4.76), 218
(4.53), 261 (4.18), 286 (4.00). FABMS m=z: 635
[M + Na]þ, HR-FABMS m=z: 635.1924 (Calcd. for
C28H36O15Na: 635.1952). For 13C and 1H NMR spectra,
24
1
see Tables 1 and 2. H NMR spectrum of the aglycone
Compound 19: Amorphous powder. ½aꢀD –43°
MeOH
max
moiety: d 7.04 (1H, d, J ¼ 9:0 Hz, H-6), 6.52 (1H, d,
J ¼ 2:5 Hz, H-3), 6.33 (1H, dd, J ¼ 9:0, 2.5 Hz, H-5),
3.80 (3H, s, –OMe), 3.68 (3H, s, AOMe).
(MeOH; c 0.49). UV k
nm (log e): 203 (4.99), 219
(4.56), 262 (4.15), 280 (sh). FABMS m=z: 711 [M + Na]þ,
HR-FABMS m=z: 711.2609 (Calcd. for C35H44O14Na:
711.2629). 13C NMR (MeOH-d4 at 35 °C): d 167.7 (C-
100), 155.0 (C-500), 150.5 (C-3), 150.2 (C-400), 145.9 (C-4),
145.6 (C-40), 137.4 (C-1), 133.8 (C-10), 125.1 (C- 700),
123.7 (C-200), 122.8, 122.6 (C-6, -60), 117.7 (C-5), 115.8
(C-50), 114.5 (C-2), 113.9 (C-300), 113.4 (C-20), 112.1 (C-
600), 102.9 (CGlc-1), 77.8 (CGlc-3), 75.6 (CGlc-5), 75.0
(CGlc-2), 72.1 (CGlc-4), 65.2 (CGlc-6), 62.2 ꢁ 2 (C-9, -90),
22
Compound 15: Amorphous powder. ½aꢀD –61°
max
MeOH
(MeOH; c 0.79). UV k
nm (log e): 203 (5.07), 212
(sh), 259 (4.48), 289 (sh). FABMS m=z: 755 [M + Na]þ,
HR-FABMS m=z: 755.2161 (Calcd. for C35H40O17Na:
1
755.2163). For 13C and H NMR spectra, see Tables 1
and 2. 1H NMR spectrum of the aglycone moiety: d 7.85
(2H, br d, J ¼ 8:5 Hz, H-30, -70), 7.12 (1H, d, J ¼ 8:0 Hz,

2010
T. Warashina et al. / Phytochemistry 65 (2004) 2003–2011
56.6 ꢁ 3, 56.4 (C-3–OMe, -30–OMe, -400–OMe, -500–
compound 8a. HPLC conditions: column; YMC-ODS
4.6 mm ꢁ 25 cm; flow rate: 1.0 ml/min. 20% MeCN; Rt,
3,4-dimethoxyphenol 10.0 min. The H2O layer was
passed through an Amberlite IRA-60E column. The
eluate was concentrated to dryness and the residue was
1
OMe), 44.1 ꢁ 2 (C-8, -80), 36.2 ꢁ 2 (C-7, -70). H NMR:
1
shown in Table 2. The H NMR spectral data of the
aglycone moiety (MeOH-d4 at 35 °C): d 6.90 (1H, d,
J ¼ 8:0 Hz, H-5), 6.65 (1H, d, J ¼ 2:0 Hz, H-2), 6.65
(1H, d, J ¼ 8:0 Hz, H-50), 6.53 (1H, br s, H-20), 6.52 (1H,
dd, J ¼ 8:0, 2.0 Hz, H-60), 6.33 (1H, dd, J ¼ 8:0, 2.0 Hz,
H-6), 3.74 (3H, s, C-3–OMe), 3.70 (3H, s, C-30–OMe),
3.57 (4H, d, J ¼ 5:0Hz, H-9, -90), 2.64 (1H, dd, J ¼ 13:5,
6.5 Hz, H-7), 2.63 (1H, dd, J ¼ 13:5, 6.5 Hz, H-70), 2.53
(1H, dd, J ¼ 13:5, 8.0 Hz, H-7), 2.52 (1H, dd, J ¼ 13:5,
8.0 Hz, H-70), 1.84 (2H, m, H-8, -80).
stirred with
D-cysteine methyl ester hydrochloride,
hexamethyldisilazane and trimethylsilylchloride in pyri-
dine using the same procedures as in previous reports
(Hara et al., 1987; Zhang et al., 1996). After the reac-
tions, the supernatant was subjected to GC analysis. GC
conditions: column; GL capillary column TC-1 (GL
Science, Inc.) 0.25 mm ꢁ 30 m, Carrier gas N2, column
temperature 230 °C; Rt,
D-glucose 21.6 min, L-glucose
20.8 min. D-glucose was detected from compound 8a.
3.3. Alkaline hydrolysis of compound 3
On the other hand, 8a being dissolved in dioxane and 2
M HCl was heated at 100 °C for 5 min. The procedures
following hydrolysis were the same as described above.
The neutralized H2O layer was reduced with NaBH4 (ca. 1
mg) for 1 h at room temperature. The procedures to al-
ditol acetate were described in previous paper (Nagatani
et al., 2001). Apiitol acetate was detected from 8a by GC
analysis. GC conditions: column; Supelco SP-2380TM
capillary column 0.25 mm ꢁ 30 m, Carrier gas N2, column
temperature 250 °C; Rt, apiitol acetate 8.1 min.
Compound 3 (1 mg) in 0.05 M NaOH (100 ll) was
treated for 3 h at room temperature with stirring under a
N2 gas atmosphere. The reaction mixture was passed
through an Amberlite IR-120B column and the eluate
was concentrated to dryness. The residue was parti-
tioned between EtOAc and H2O. Both layers were
concentrated to dryness, and by HPLC analysis, 3,4,5-
trimethoxybenzoic acid and ajugol (1a) were, respec-
tively, identified on comparison with authentic samples.
HPLC conditions: column; YMC-ODS 4.6 mm ꢁ 25 cm;
flow rate: 1.0 ml/min. 22.5% MeCN + 0.05% trifluoro-
acetic acid (TFA); Rt, 3,4,5-trimethoxybenzoic acid 16.6
min. 5% MeCN; Rt, ajugol 17.4 min.
24
Compound 8a: Amorphous powder. ½aꢀD –113°
MeOH
max
(MeOH; c 0.52,). UV k
nm (log e): 202 (4.48), 223
(3.92), 281 (3.53). FABMS m=z: 471 [M + Na]þ, HR-
FABMS m=z: 471.1475 (Calcd. for C19H28O12Na:
471.1478). 13C NMR (MeOH-d4 at 35 °C): d 153.9 (C-1),
151.2 (C-3), 146.3 (C-4), 114.2 (C-5), 111.0 (CApi-1),
109.6 (C-6), 104.5 (C-2), 103.5 (CGlc-1), 80.5 (CApi-3),
78.0 ꢁ 2 (CGlc-3, CApi-2), 77.0 (CGlc-5), 75.0 ꢁ 2 (CGlc-2,
CApi-4), 71.7 (CGlc-4), 68.8 (CGlc-6), 65.6 (CApi-5), 57.2,
3.4. Alkaline hydrolysis of compounds 5, 8 and 11
Compounds 5, 8 and 11 (ca. 10 mg) were dissolved in
0.05 M NaOH (1 ml) and stirred for 2–3 h at room
temperature with stirring under a N2 gas atmosphere.
The reaction mixture was treated the above same way.
HPLC analysis of the residue from each EtOAc layer
suggested that 3,4-dimethoxybenzoic acid was produced
from 5 and 4-methoxybenzoic acid was yielded from 8
and 11. HPLC conditions: column; YMC-ODS 4.6
mm ꢁ 25 cm; flow rate: 1.0 ml/min. 20% MeCN + 0.05%
TFA; Rt, 3,4-dimethoxybenzoic acid 15.6 min. 27.5%
MeCN + 0.05% TFA; Rt, 4-methoxybenzoic acid 12.4
min. Purification of the residue from each H2O layer
using HPLC (YMC-ODS 10 mm ꢁ 25 cm, 7.5% MeCN
in water) afforded osmanthuside H (5a, 5 mg), 8a (6 mg)
1
56.6 (C-3–OMe, -4–OMe). H NMR (MeOH-d4 at 35
°C): d 6.87 (1H, d, J ¼ 8:5 Hz, H-5), 6.78 (1H, d, J ¼ 2:5
Hz, H-2), 6.69 (1H, dd, J ¼ 8:5, 2.5 Hz, H-6), 4.98 (1H,
d, J ¼ 2:0 Hz, HApi-1), 4.74 (overlapping, HGlc-1), 4.02
(1H, dd, J ¼ 11:5, 2.0 Hz, HGlc-6), 3.96 (1H, d, J ¼ 10:0
Hz, HApi-4), 3.90 (1H, d, J ¼ 2:0 Hz, HApi-2), 3.82 (3H, s,
C-3–OMe), 3.78 (3H, s, C-4–OMe), 3.75 (1H, d, J ¼ 10:0
Hz, HApi-4), 3.62 (1H, dd, J ¼ 11:5, 6.5 Hz, HGlc-6), 3.57
(2H, s, HApi-5), 3.55 (1H, m, HGlc-5), 3.42 (overlapping,
H
Glc-2, HGlc-3), 3.33 (overlapping, HGlc-4).
3.5. Acid hydrolysis of compound 16
and 3,4,5-trimethoxyphenyl 1-O-b-D-apiofuranosyl-
(1 ! 6)-b-
D
-glucopyranoside (11a, 5 mg). Compounds
Compound 16 (10 mg) was dissolved in 0.2 M HCl (750
ll) and dioxane (300 ll), and heated at 95 °C for 5 h. The
reaction mixture was diluted with H2O and extracted with
EtOAc. The EtOAc layer was dried (Na2SO4) overnight,
the Na2SO4 removed by filtration, and the EtOAc evap-
orated in vacuo to give a residue. The latter was subjected
to HPLC (YMC-ODS 10 mm ꢁ 25 cm, 27.5% MeCN in
water), to afford ())-6-hydroxymellein 16a (0.5 mg); the
latter being identified on the basis of its 1H NMR
(Breinholt et al., 1993), CD spectral data (Antus et al.,
5a and 11a were identified on comparison of the 1H
and 13C NMR spectral data with the reported ones
(Sugiyama and Kikuchi, 1993; Kanchanapoom et al.,
2002).
Compound 8a (ca. 0.5 mg) was dissolved in dioxane
and 2 M HCl (50 ll each). The solution was heated at
100 °C for 1 h, and the reaction mixture was partitioned
between EtOAc and H2O. By HPLC analysis, 3,4-di-
methoxyphenol was detected from the EtOAc layer of

T. Warashina et al. / Phytochemistry 65 (2004) 2003–2011
2011
1983; Venkatasubbaiah and Chilton, 1991) and optical
rotation value (Breinholt et al., 1993).
tected from 9 and 10, and 3,4,5-trimethoxyphenyl 1-O-b-
-apiofuranosyl-(1 ! 6)-b- -glucopyranoside (11a) and
secoisolariciresinol 4-O-b- -glucopyranoside (18a) were
from 12 and 19, respectively. HPLC conditions: column;
YMC-ODS 4.6 mm ꢁ 25 cm; flow rate: 1.0 ml/min. 10%
MeCN in water; Rt, 5a 10.4 min, 8a 10.2 min, 11a 15.6
min, 15% MeCN in water; Rt, 18a 14.4 min.
The residues of H2O layer of compounds 13–17 were
hydrolyzed with 2 M HCl and dioxane (50 ll each).
The following procedures were described above. By
HPLC analysis, 4-methoxyphenol, vanillyl alcohol and
())-6-hydroxymellein were detected from the residues
of the EtOAc layer of compounds 13, 15 and 17,
respectively. YMC-ODS 4.6 mm ꢁ 25 c; flow rate: 1.0
ml/min. 17.5% MeCN in water; Rt, 4-methoxyphenol
17.2 min, 10% MeCN in water; Rt, vanillyl alcohol 10.4
min, 27.5% MeCN in water; Rt, ())-6-hydroxymellein
D
D
24
22
Compound 16a: ½aꢀD – 33° (MeOH; c 0.05)(lit. ½aꢀD
D
1
–57° (MeOH; c 0.07)). H NMR (CDCl3): d 11.20 (1H,
br s, -OH), 6.31 (1H, br d, J ¼ 2:0 Hz, H-5 or -7), 6.20
(1H, br s, H-5 or -7), 4.67 (1H, m, H-3), 2.86 (2H,
overlapping, H-4), 1.51 (3H, d, J ¼ 6:5 Hz, H-11).
CDDe (nm): –2.0 (232), +0.24 (248), )1.4 (268), +0.13
(302) (MeOH; c .05)(lit. CD De (nm): )2.4 (195), )2.64
(232), +0.83 (246), )2.96 (268), +0.39 (302) (MeCN)).
3.6. Alkaline hydrolysis of compound 18
Compound 18 (ca. 5 mg) was dissolved in 0.1 M
NaOH (1.4 ml) and stirred for 4.5 h at room tempera-
ture with stirring under N2. The reaction mixture was
passed through an Amberlite IR-120B column with the
eluate concentrated to dryness. The residue was parti-
tioned between EtOAc and H2O. Both layers were
concentrated to dryness, with purification of this solu-
bles HPLC (YMC-ODS 10 mm ꢁ 25 cm, 15% MeCN in
15.6 min. D-glucose and apiitol acetate were detected
from compounds 13, 15 and 17 by GC analysis.
(Conditions of GC analysis were described above.)
water) to afford secoisolariciresinol 4-O-b-
D
1
-glucopyr-
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25
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droxyphenyl)ethyl 1-O-b-
D
-apiofuranosyl-(1 ! 6)-b-
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glucopyranoside (5a) was identified from the residues of
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the H2O layer of compounds 6 and 7 by HPLC analysis.
In the same way, 3,4-dimethoxyphenyl 1-O-b-
D-apio-
furanosyl-(1 ! 6)-b- -glucopyranoside (8a) was de-
D