Steroidal glycosides from the aerial part of Asclepias incarnata

Article abstract of DOI:10.1016/S0031-9422(99)00560-9

The aerial part of Asclepias incarnata afforded 34 pregnane glycosides. These were confirmed to have lineolon, isolineolon, ikemagenin, 12-O- nicotinoyllineolon, deacylmetaplexigenin, metaplexigenin, rostratamine, 12-O- acetyllineolon, 15β-hydroxylineolon

Full text of DOI:10.1016/S0031-9422(99)00560-9

Phytochemistry 53 (2000) 485±498
www.elsevier.com/locate/phytochem
Steroidal glycosides from the aerial part of Asclepias incarnata
Tsutomu Warashina*, Tadataka Noro
Institute for Environmental Sciences, University of Shizuoka, 52-1, Yada, Shizuoka, 422-8526, Japan
Received 15 June 1999; received in revised form 10 September 1999
Abstract
The aerial part of Asclepias incarnata a€orded 34 pregnane glycosides. These were con®rmed to have lineolon, isolineolon,
ikemagenin, 12-O-nicotinoyllineolon, deacylmetaplexigenin, metaplexigenin, rostratamine, 12-O-acetyllineolon, 15b-
hydroxylineolon and 15b-hydroxyisolineolon moieties as their aglycones, and 2,6-dideoxyhexopyranose, glucopyranose and
allopyranose as the corresponding sugar constituents. Their structures were determined using both spectroscopic and chemical
methods. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Asclepias incarnata; Asclepiadaceae; Pregnane glycoside; 2,6-Dideoxyhexopyranose
1. Introduction
To determine the component sugars, a mixture of
pregnane glycosides was subjected to acid hydrolysis.
The acquired component sugars were fractionated by
silica gel column chromatography, and cymarose,
oleandrose and digitoxose were obtained (See chart 2).
The absolute con®gurations of cymarose, oleandrose
and digitoxose were believed to be in the D-form based
on their optical rotation values (Tsukamoto, Hayashi,
Kaneko & Mitsuhashi, 1986; Nakagawa, Hayashi,
Wada & Mitsuhashi, 1983; Abe, Mohri, Okabe &
Yamauchi, 1994).
In connection with a study on the constituents of
plants in the Asclepiadaceae family, we have investi-
gated the aerial part of Asclepias incarnata L. The pre-
sent paper describes the isolation and structural
elucidation of 34 new oxypregnane glycosides.
2. Results and discussion
Glucose in the sugar chain of each compound was
deduced to be in the D-form based on the reaction
with D-cysteine methyl ester hydrochloride followed by
GC analysis (see Section 3).
The MeOH extract obtained from the dried aerial
part of A. incarnata L. (1.3 kg) was suspended in
water. The suspension was then extracted with diethyl
ether and partitioned into an ether layer and a H2O
layer. The H2O layer was directly passed through a
Mitsubishi Diaion HP-20 column and the adsorbed
material was subsequently eluted with 50% MeOH in
water, 70% MeOH in water, and MeOH. The Et2O
layer and MeOH eluates of the HP-20 column were
concentrated, and the residues were rechromato-
graphed on a silica gel column and on semi-prepara-
tive HPLC, respectively, to give oxypregnane
glycosides (1±34).
Compound 1 was suggested to have the molecular
formula, C42H66O16, based on FABMS [positive
FABMS ion at m/z 827 [M + H]+]. This molecular
formula was supported by the 13C-NMR spectrum, in
which 42 carbon signals were observed and these sig-
nals consisted of eight methyl carbon signals, 10 meth-
ylene carbon ones, 16 methine carbon ones and eight
quaternary carbon ones. In the 13C-NMR spectrum of
1, the signals due to the aglycone moiety were in good
agreement with those due to metaplexigenin (39) (Mit-
suhashi & Nomura, 1965) within the range of glycosy-
lation shifts at C-3 (+6.1 ppm), C-2 ( 2.2 ppm) and
* Corresponding author.
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00560-9

486
T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
C-4 ( 4.0 ppm). In the 1H- and 13C-NMR spectra,
three anomeric proton and carbon signals were
observed at d 5.46, 5.39, 4.79 and d 96.4, 99.8, 101.6.
Thus, compound 1 was believed to be metaplexigenin
3-O-trioside. On acid hydrolysis of 1 with 0.05 N HCl,
metaplexigenin, digitoxose and oleandrose were
obtained as the component aglycone and sugars. These
sugars were identi®ed as b-D-digitoxopyranose and b-
D-oleandropyranose, as judged from the coupling con-
stant of each anomeric proton signal ꢀJ ˆ 9:5, 2.0 Hz).
Furthermore, the presence of the characteristic H-3
signals of digitoxose ‰d 4.63 (1H, br s ) and 4.66 (1H,
br s)] and one methoxyl proton signal ‰d 3.46 (3H, s)]
in the 1H-NMR spectrum suggested that the sugar
moiety included two digitoxopyranose and one olean-
dropyranose. The 1H±1H COSY experiment allowed
the sequential assignments of the proton signals for
each sugar as shown in Table 3, starting from each
anomeric proton signal. For the sugar linkage, the
di€erence nuclear Overhauser e€ect (NOE) spectra
were measured on irradiation of the anomeric proton
signals, and NOEs were observed as follows, d 5.46
(1H, dd, J = 9.5, 2.0 Hz, H-1' of b-D-digitoxopyra-
nose) and d 3.85 (1H, m, H-3 of the aglycone), d 5.39
(1H, dd, J = 9.5, 2.0 Hz, H-10 of b-D-digitoxopyra-
nose) and d 3.50 (1H, dd, J = 9.5, 3.0 Hz, H-4' of b-
D-digitoxopyranose), and d 4.79 (1H, dd, J = 9.5, 2.0
Hz, H-1'0 of b-D-oleandropyranose) and d 3.47 (1H,
dd, J = 9.5, 3.0 Hz, H-40 of b-D-digitoxopyranose).
Hence, the structure of 1 was established as metaplexi-
1H±1H COSY spectrum. Determination of the sugar
sequence was carried out by the results of the di€er-
ence NOE experiment irradiating the anomeric proton
signals. NOEs were observed between d 4.92 (H-1' of
b-D-digitoxopyranose)/3.57 (H-3 of the aglycone); 4.88
(H-10 of b-D-digitoxopyranose)/3.22 (H-4' of b-D-digi-
toxopyranose); 4.90 (H-1'0 of b-D-digitoxopyranose)/
3.20 (H-40 of b-D-digitoxopyranose); 4.55 (H-100 of b-
D-oleandropyranose)/3.20 (H-4'0 of b-D-digitoxopyra-
3
nose). Furthermore, the JCOCHS were detected as fol-
lows in the 1H-detected heteronuclear multiple-bond
connectivity (HMBC) spectrum, d 95.9 (C-1' of b-D-
digitoxopyranose) and 3.57 (H-3 of the aglycone); d
98.3 (C-10 of b-D-digitoxopyranose) and 3.22 (H-4' of
b-D-digitoxopyranose); d 98.3 (C-3'0 of b-D-digitoxo-
pyranose) and 3.20 (H-40 of b-D-digitoxopyranose);
d 100.4 (C-100 of b-D-oleandropyranoside) and 3.20
(H-4'0 of b-D-digitoxopyranose). Based on the above
evidence, the structure of 2 was determined to be meta-
plexigenin 3-O-b-D-oleandropyranosyl-(1 4 4)-b-D-
digitoxopyranosyl-(1 4 4)-b-D-digitoxopyranosyl-(1 4
4)-b-D-digitoxopyranoside.
The molecular formulae of compounds 3±10 were
C55H80O18,
C46H74O17,
C46H74O17,
C48H76O18,
C52H77NO18, C46H74O18, C52H77NO19 and C46H74O18,
respectively, based on observation of [M + H]+ and/
or [M + Na]+ ion peaks in FABMS and comparison
of each H- and 13C-NMR spectral data with those of
1
2 and each aglycone. On acid hydrolysis, 3±10 a€orded
lineolon (35) (Yamagishi, Hayashi, Mitsuhashi, Ina-
mori & Matsushita, 1973a, 1973b; Warashina & Noro,
1994), isolineolon (36) (Yamagishi et al., 1973a, Yama-
gishi et al., 1973b), 37, ikemagenin (Abe et al., 1994;
Yamagishi & Mitsuhashi, 1972), 12-O-nicotinoyllineo-
lon (Warashina & Noro, 1996), deacylmetaplexigenin
(38)(Tsukamoto, Hayashi & Mitsuhashi, 1985; Mitsu-
hashi & Nomura, 1965), rostratamine (Gellert & Sum-
mons, 1973) and 40 as the aglycone moiety,
respectively, and digitoxose and oleandrose as the
genin
pD-digitoxopyranosyl-(1 4 4)-b-D-digitoxopyranoside.
Compound had the molecular formula,
3-O-b-D-oleandropyranosyl-(1
4
4)-b-
2
C48H76O19, by observating of [M + H]+ ion peaks on
FABMS and the consequences of the 1H-, 13C-NMR
spectra and the 1H-detected heteronuclear multiple
quantum coherence (HMQC) experiment. On acid hy-
drolysis, 2 a€orded metaplexigenin (39) as the agly-
cone moiety, and digitoxose and oleandrose were
obtained as the component sugars. In the H- and 13C-
sugar moiety. Because of the consistency of their H-
1
1
NMR spectra which was measured in CDCl3 solution,
four anomeric proton and carbon signals were shown
at d 4.92 (1H, dd, J = 9.5, 2.0 Hz), 4.88 (1H, dd, J =
9.5, 2.0 Hz), 4.90 (1H, dd, J = 9.5, 2.0 Hz), 4.55 (1H,
dd, J = 9.5, 2.0 Hz) and d 95.9, 98.3 Â 2, 100.4. The
13C-NMR spectral comparison of 2 with that of meta-
plexigenin showed glycosylation shifts were for the C-
3, C-2 and C-4 signals (in pyridine-d5 solution). Thus,
compound 2 was considered as metaplexigenin 3-O-tet-
and 13C-NMR spectral data with those of 2, it was
shown that 3±10 possessed the same sugar sequences
in the oligosaccharide moiety as that of 2, with the
sugar moiety attached to the C-3 position of each agly-
cone. Thus, structures of 3±10 were elucidated as
shown.
Compounds 11±15 had the molecular formulae,
C52H84O22, C52H84O22, C52H86O23, C54H86O24 and
1
C52H84O23, respectively, based on FABMS and the H-
and 13C-NMR spectra. On acid hydrolysis with 0.05 N
HCl and the 1H- and 13C-NMR spectra, 11±15 were
determined to be pregnane 3-O-pentaosides whose
aglycones were lineolon (35) for 11, isolineolon (36)
for 12, 37 for 13, metaplexigenin (39) for 14 and 40
for 15. The sugar sequences of these compounds were
the same based on the 1H- and 13C-NMR spectral
1
raoside. As the H-NMR spectrum of 2 exhibited one
methoxyl proton signal at d 3.40 (3H, s), the sugar
sequence of 2 consisted of three digitoxopyranose and
one oleandropyranose moiety. The coupling constant
of each sugar indicated that these sugars had a b-gly-
cosidic linkage. The proton signals for each sugar were
assigned as shown in Table 4 on the basis of the

T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
487
data. On acid hydrolysis of the above, digitoxose,
oleandrose and glucose were liberated with each agly-
cone. In the H-NMR spectrum of 13, the character-
ides retained b-glycosidic linkage as shown by the
coupling constants of the H-1 signals. In the H-NMR
1
1
spectrum of 19, the characteristic H-3 signals of cym-
aropyranose and digitoxopyranose were observed at d
4.09 (1H, q, J = 3.0 Hz), 4.63 (1H, br s ) Â 2, and two
methoxyl proton signals were observed at d 3.46 (3H,
s ) and 3.62 (3H, s). The signals of the terminal olean-
drose were also observed in the 13C-NMR spectrum.
Therefore, one methoxyl proton signal at d 3.46
belonged to C-3-OMe of b-D-oleandropyranose and
another methoxyl signal at d 3.62 was due to C-3-OMe
of b-D-cymaropyranose. Based on analysis of the
HMQC and HMBC spectra, the methoxyl proton sig-
istic H-3 signals of digitoxopyranose were observed at
d 4.58 (1H, q, J = 3.0 Hz), 4.62 (1H, q, J = 3.0 Hz)
and 4.63 (1H, q, J = 3.0 Hz) and one methoxyl signal
of oleandropyranose was observed at d 3.52 (3H, s).
Thus, the oligosaccharide moieties of 11±15 were com-
posed of three digitoxopyranose, one oleandropyra-
nose and one glucopyranose whose glycosidic linkages
were deduced to be in a b-orientation based on the
coupling constants of the anomeric proton signals.
Enzymatic hydrolysis of 13 with cellulase produced
compound 5, which was con®rmed by comparison of
the 1H-NMR spectral data and HPLC analysis with
data of an authentic sample. Moreover, in the di€er-
ence NOE experiment of 13, irradiation of the anome-
ric proton signal of glucopyranose at d 5.10 (1H, d, J
= 8.0 Hz) caused enhancement of the signal intensity
at d 3.63 (H-400 of b-D-oleandropyranose). So, b-D-glu-
copyranose was attached to the C-4 position of b-D-
oleandropyranose, and the sugar sequence of 13 was
decided as 3-O-b-D-glucopyranosyl-(1 4 4)-b-D-olean-
dropyranosyl-(1 4 4)-b-D-digitoxopyranosyl-(1 4 4)-b-
D-digitoxopyranosyl-(1 4 4)-b-D-digitoxopyranoside.
Similarly, the structures of compounds 11, 12, 14 and
15 were elucidated as shown.
3
nal at d 3.62 showed a JHCOC to the C-3 signal at d
1
78.1, and this C-3 signal had a JCH to the signal at d
4.09. The carbon and proton signals at d 78.1 and 4.09
were assigned to C-3 and H-3 of b-D-cymaropyranose;
the remaining signals at d 4.63 Â 2 corresponded to H-
1
3 of b-D-digitoxopyranose. Using the H±1H COSY ex-
periment, the assignments of the proton signals in the
oligosaccharide moiety were carried out as shown in
Table 3. In the di€erence NOE experiment, upon ir-
radiation of the anomeric proton signals, NOEs were
observed as follows: d 5.26 (H-1' of b-D-cymaropyra-
nose) and d 3.82 (H-3 of the aglycone); d 5.32 (H-10 of
b-D-digitoxopyranose) and d 3.52 (H-4' of b-D-cymaro-
pyranose); d 5.38 (H-1'0 of b-D-digitoxopyranose) and
d 3.48 (H-40 of b-D-digitoxopyranose); d 4.80 (H-100 of
b-D-oleandropyranose) and d 3.48 (H-4'0 of b-D-digi-
toxopyranose). Consequently, the sugar linkage of 19
was found to be 3-O-b-D-oleandropyranosyl-(1 4 4)-b-
D-digitoxopyranosyl-(1 4 4)-b-D-digitoxopyranosyl-(1
4 4)-b-D-cymaropyranoside. This sugar linkage was
supported by a long-range correlation between the
anomeric carbon signals and the H-4 signals of mono-
saccharides in the HMBC spectrum. Similarly, the
structures of 17, 18, 20±23 and 24 were established as
shown.
Compound 16 had the molecular formula,
C52H84O23, based on FABMS and the 1H- and 13C-
NMR spectra. As the H- and 13C-NMR spectra were
1
almost identical to those of 15, the structure of 16 was
considered to be similar to that of 15, but the signals
due to an unidenti®ed sugar appeared instead of the
signals due to the terminal b-D-glucopyranose. On acid
hydrolysis of 16, D-allose was obtained, together with
digitoxose and oleandrose as the sugar moiety (see Sec-
tion 3). Therefore, this unidenti®ed sugar was deter-
mined to be b-D-allopyranose in consideration of the
coupling constant of the H-1 signal, and the structure
of 16 was elucidated as presented.
Compounds 25±29 had the molecular formulae,
C53H86O22, C53H86O22, C55H88O23, C59H89NO23 and
C55H88O24, respectively, based on the results of
The molecular formulae of compounds 17±24 were
suggested to be C47H76O17, C47H76O17, C49H78O18,
C56H82O18, C53H79NO18, C49H78O19, C47H76O18 and
FABMS, the H- and 13C-NMR spectra. Analyzing the
1
1H- and 13C-NMR spectral data, 25±29 were found to
be pregnane 3-O-pentaosides with identical sugar
sequences. On acid hydrolysis of 25±29, cymarose,
digitoxose, oleandrose and glucose were shown to con-
stitute the sugar moieties, and the aglycones were
determined to be lineolon (35) for 25, isolineolon (36)
for 26, 37 for 27, 12-O-nicotinoyllineolon for 28 and
metaplexigenin (39) for 29. Compounds 25±29 dis-
played the signals of a terminal b-D-glucopyranose as
1
C47H76O18, respectively, based on FABMS and the H-
and 13C-NMR spectra. From the results of acid hy-
drolysis and the H- and 13C-NMR spectral data, these
1
compounds were identi®ed to be pregnane 3-O-tetrao-
sides whose aglycones were lineolon (35) for 17, isoli-
neolon (36) for 18, 37 for 19, ikemagenin for 20, 12-O-
nicotinoyllineolon for 21, metaplexigenin (39) for 22,
40 for 23 and 41 for 24. Compounds 17±24 possessed
the same sugar sequences based on the 1H- and 13C-
NMR spectral data of the oligosaccharide moieties.
On acid hydrolysis, compounds 17±24 yielded cymar-
ose, digitoxose and oleandrose as the constituents of
each oligosaccharide moiety, and these monosacchar-
did 11±15, as shown by the H- and 13C-NMR spectra.
1
Enzymatic hydrolysis with cellulase produced 17 from
25, 18 from 26, 19 from 27, 21 from 28 and 22 from
29. The di€erence NOE spectrum of 28 showed, upon
irradiation of the H-1 signal of b-D-glucopyranose ‰d

488
T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
Chart 1

T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
489
5.10 (1H, d, J = 8.0 Hz)], an NOE to the H-4 signal
of b-D-oleandropyranose ‰d 3.62 (overlapping with
other signals)]. Accordingly, it was concluded that the
b-D-glucopyranose was attached to the C-4 position of
b-D-oleandropyranose. Thus, the structures of 25±29
were elucidated.
By observation of the [M + H]+ ion peak in
FABMS, the molecular formulae of compounds 30±32
were deduced to be C48H78O17, C48H78O17 and
C50H80O18, respectively. On acid hydrolysis of 30±32,
cymarose, digitoxose and oleandrose were obtained
along with the aglycones lineolon (35), isolineolon (36)
and 37, respectively. As evident from the 1H-NMR
spectra, each sugar moiety was composed of two b-D-
cymaropyranose, one b-D-digitoxopyranose and other
b-D-oleandropyranose. Their sugar sequences were
determined to be 3-O-b-D-oleandropyanosyl-(1 4 4)-b-
D-digitoxopyranosyl-(1 4 4)-b-D-cymaropyranosyl-(1
4 4)-b-D-cymaropyranoside, using the same di€erence
NOE procedure used for 31, i.e. irradiation of the H-1
signal of each monosaccharide. Thus, the structures of
30±32 were elucidated as shown.
Compound 33 was presumed to be a pregnane 3-O-
pentaoside based on the 1H- and 13C-NMR spectral
data. Compound 33 was hydrolyzed to yield 40, cym-
arose, digitoxose, oleandrose and glucose, and com-
parison of the NMR spectral data with those of 15
suggested that b-D-glucopyranose was the terminal
sugar in the oligosaccharide sequence. The 1H-NMR
spectral data of the oligosaccharide sequence of the
product of enzymatic hydrolysis of 33 were consistent
with those of 30±32. Therefore, the sugar sequence of
33 was determined to be 3-O-b-D-glucopyranosyl-(1 4
4)-b-D-oleandropyranosyl-(1 4 4)-b-D-digitoxopyrano-
syl-(1 4 4)-b-D-cymaropyranosyl-(1 4 4)-b-D-cymaro-
pyranoside.
Fig. 1. Observed NOE correlations in compounds 40 and 41.
mula, C23H34O6, based on the FABMS spectrum.
(Positive FABMS ion peak at m/z 407 [M + H]+).
1
This molecular formula was supported by the H- and
13C-NMR spectra, in which the signals of an acetyl
group were observed at d 2.05 (3H, s, COCHÃ3) and d
167.0 (CÃOCH3), 20.8 (COCÃH3), in addition to the
signals of lineolon. On comparing the NMR spectral
data of 37 with those of lineolon, acylation shifts were
displayed at C-11 ( 4.6 ppm), C-12 (+4.2 ppm) and
C-13 ( 2.3 ppm). Furthermore, the H-12 signal was
shifted down®eld to d 5.05. These facts demonstrated
that the acetyl group was attached to C-12-OH of
lineolon, and 37 was determined to be 12-O-
acetyllineolon.
Compound 34 was presumed to be a pregnane 3-O-
tetraoside, with the molecular formula C49H80O18.
Acid hydrolysis of 34 a€orded 40 as the aglycone moi-
ety and cymarose and oleandrose as the sugar moi-
eties. The H- and 13C-NMR spectral data of 34 were
1
in good agreement with those of calotroposide E (Shi-
buya, Zhang, Park, Beak & Takeda, 1992). The sugar
sequence of 34 was determined to be 3-O-b-D-olean-
dropyranosyl-(1 4 4)-b-D-oleandropyranosyl-(1 4 4 )-
b-D-cymaropyranosyl-(1 4 4)-b-D-cymaropyranoside;
therefore, the structure of 34 was determined as shown
in Chart 1.
In the FABMS spectrum, compound 40 showed an
[M + H]+ ion peak at m/z 381, 16 mass units larger
than that of lineolon, suggesting the molecular formula
C21H32O6. Upon comparison of the 13C-NMR spectral
data of 40 with that of lineolon, a new carbinyl carbon
signal was observed at d 71.7 instead of a methylene
carbon signal. In the HMQC spectrum, this carbinyl
The aglycones obtained by acid hydrolysis of a mix-
ture of pregnane glycosides together with sugars were
identi®ed as lineolon (35), isolineolon (36), deacylmeta-
1
plexigenin (38), metaplexigenin (39) based on the H-
and 13C-NMR spectral data and ‰aŠ values. In ad-
1
carbon signal showed JCH to d 4.54 (1H, m ), and in
D
3
2
the HMBC spectrum, JCCCHS and a JCCH were
observed d 59.4 (C-17) and d 4.54; d 71.7 and d 5.07
(1H, s, 14-OHÃ); d 71.7 and d 3.33 (1H, dt, J = 14.5,
dition, one new acylated pregnane (37) and two new
oxypregnanes (40 and 41) were obtained.
Compound 37 was presumed to have molecular for-

490
T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
10.0 Hz, H-16). Thus, this carbinyl carbon and proton
signals were due to C-15 and H-15, and the hydroxyl
group was attached to C-15. The orientation of the hy-
droxyl group was presumed to be b, based on com-
parison of the C-15 signal to that of 17-epi-
hancogenine (Konda, Toda & Harigaya, 1992). This
was proved using a di€erence NOE experiment,
wherein irradiation at d 4.03 (1H, dd, J = 12.0, 4.0
Hz, H-12) and d 1.67 (1H, dd, J = 13.5, 2.5 Hz, H-9)
showed NOEs to d 4.54 (1H, m, H-15), and irradiation
at d 4.54 (H-15) exhibited NOEs to d 4.03 (H-12) and
1.67 (H-9) (see ®g. 1). Thus, the orientation of H-15
was decided to be a, and in conclusion, this hydroxyl
group had b-orientation. Based on the above argu-
ments, the structure of 40 was elucidated as 15b-
hydroxylineolon.
3000 gas chromatograph, and HPLC analyses
employed a JASCO 800 system instrument.
3.1. Plant material
Asclepias incarnata L. was cultivated in the botanical
garden and harvested on July 1996 in Shizuoka, Japan
and identi®ed by Professor Noro (University of Shi-
zuoka).
3.2. Extraction and isolation
The air dried aerial part of A. incarnata L. (1.3 kg)
was extracted twice with MeOH, heated until re¯ux.
The resulting extracts were combined, then concen-
trated under reduced pressure with the resulting resi-
due next suspended in H2O (4 l). This suspension was
extracted with Et2O (1.5 l). The H2O layer was directly
passed through a Mitsubishi Diaion HP-20 column
and the adsorbed material was subsequently eluted
with 50% MeOH in water, 70% MeOH in water, and
MeOH. The Et2O layer and MeOH eluates of the HP-
20 column were concentrated, respectively, and each
residue was rechromatographed on a silica gel column
with CHCl3±MeOH as eluant system, and on semi-pre-
parative HPLC (Develosil-ODS, PhA., C-8 and YMC-
ODS, eluted with MeCN±H2O (22.5±42.5% MeCN in
water) and/or MeOH±H2O (55±80% MeOH in water)
systems to give compounds 1 (18 mg), 2 (37 mg), 3 (12
mg), 4 (15 mg), 5 (12 mg), 6 (5 mg), 7 (5 mg), 8 (20
mg), 9 (6 mg), 10 (30 mg), 11 (17 mg), 12 (11 mg), 13
(15 mg), 14 (10 mg), 15 (8 mg), 16 (4 mg), 17 (10 mg),
18 (15 mg), 19 (17 mg), 20 (10 mg), 21 (3 mg), 22 (15
mg), 23 (18 mg), 24 (4 mg), 25 (8 mg), 26 (9 mg), 27
(10 mg), 28 (5 mg), 29 (7 mg), 30 (5 mg), 31 (5 mg), 32
As compound 41 showed the same [M + H]+ ion
peak in FABMS spectrum as did 40, the molecular
formula of 41 was presumed to be C21H32O6. The 13C-
NMR spectrum was similar to that of 40; 41 was
deduced to have a hydroxyl group at C-15b the same
as did 40. This was con®rmed by the analysis of the
1
NMR spectra ꢀ H±1H COSY, HMQC, HMBC and
di€erence NOE). In the 13C-NMR spectrum, the C-20
signal shown at d 215.3 was in good agreement with
that of isolineolon. Thus, 41 was determined to be
15b-hydroxyisolineolon.
3. Experimental
Optical rotations were determined using a JASCO-
DIP1000 digital polarimeter. FABMS spectra were col-
lected on a JEOL JMS-SX120 spectrometer in m-nitro-
benzyl alcohol, whereas both H- and 13C-NMR were
(2 mg), 33 (6 mg) and 34 (8 mg).
1
23
D
recorded in pyridine-d5 and/or CDCl3 solutions on a
JOEL JNM A-400 (400 and 100.40 MHz, respectively)
spectrometer. Chemical shifts are given on the d (ppm)
scale with tetramethylsilane (TMS) as an internal stan-
dard. UV spectra were measured with a Beckman
UV640 spectrophotometer. GC utilised a Hitachi G-
Compound 1. Amorphous powder; ‰aŠ
9.78
(MeOH; c 1.02); FABMS m/z: 827 [M + H]+, 849
[M + Na]+; 13C- and 1H-NMR spectral data: see
Tables 1, 2 and 3.
24
D
Compound 2. Amorphous powder; ‰aŠ
4.58
(MeOH; c 1.04); FABMS m/z: 957 [M + H]+, 979 [M
Chart 2

T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
491
Table 1
13C-NMR spectral data of the aglycone moiety (in pyridine-d5 solution at 358C)
3
4
5
6
7
8
1
9
10
24
Carbon No.
C-1
C-2
39.1
30.0
77.8
39.4
139.6
119.4
34.5
74.6
45.2
37.5
29.4
69.0
58.0
87.4
35.4
22.2
61.5
14.8
18.4
210.4
32.1
39.2
30.0
77.8
39.4
139.5
119.7
37.3
74.5
45.6
37.6
28.5
74.0
56.7
86.6
36.2
24.6
58.7
11.6
18.7
216.8
32.2
39.0
29.9
77.7
39.3
139.5
119.1
34.1
74.6
44.8
37.5
24.8
73.2
55.7
87.5
35.1
21.8
60.5
15.6
18.2
209.6
32.3
39.0
29.9
77.7
39.3
139.5
119.1
34.2
74.6
44.8
37.6
25.0
73.4
55.9
87.5
35.2
22.0
60.5
15.8
18.2
209.3
32.2
39.0
29.9
77.7
39.3
139.5
119.1
34.2
74.5a
44.7
37.6
25.0
74.3a
56.1
87.5
35.1
22.2
60.2
15.8
18.2
209.8
32.4
39.1
30.0
77.7
39.4
139.5
119.4
34.2
74.3
45.0
37.4
29.4
69.0
60.5
89.3
35.1
32.8
92.6
9.4
39.0
29.9
77.7
39.3
139.4
119.1
34.7
74.3
44.6
37.4
24.8
73.6
57.9
89.4
32.9
33.7
92.4
10.4
18.2
210.1
27.6
39.0
29.9
77.6
39.3
139.4
119.1
34.7
74.3a
44.5
37.4
25.1
74.7a
58.3
89.5
33.8
33.3
92.5
10.7
18.2
210.5
27.9
39.0
30.0
77.8
39.4
138.9
119.7
35.4
74.7
45.3
37.6
29.2
68.7
58.2
86.3
71.7
34.5
59.4
15.2
18.7
209.7
32.1
39.2
30.1
77.8
39.4
138.4
120.2
35.1
74.9
45.9
37.7
28.5
73.5
56.3
84.7
74.9
36.0
56.3
12.5
18.7
215.2
31.9
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
Ester
C-1'
C-2'
C-3'
C-4'
C-5'
C-6'
C-7'
C-8'
C-9'
18.4
209.6
27.8
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
169.9
165.9
119.4
144.8
135.1
128.6
129.3
130.5
129.3
128.6
164.5
±
±
±
±
±
±
±
±
±
±
169.8
164.3
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
20.8
±
20.8
±
153.8
127.0
137.1
123.4
151.1
±
153.8
126.9
137.1
123.5
151.5
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
a Interchangeable within each column.
+ Na]+; 13C- and H-NMR spectral data: see Tables
2±4. The 13C-NMR spectral data of the aglycone moi-
NMR spectral data of the sugar moiety were in good
agreement with those of 2.
1
24
D
ety were in good agreement with those of 1.
Compound 7. Amorphous powder; ‰aŠ
19.18
24
D
Compound 3. Amorphous powder; ‰aŠ
+3.28
(MeOH; c 0.57); FABMS m/z: 1004 [M + H]+; UV
l
(MeOH; c 1.24); FABMS m/z: 899 [M + H]+, 921
[M + Na]+; 13C-NMR spectral data: see Table 1. The
13C- and 1H-NMR spectral data of the sugar moiety
nm ꢀlog e): 219 (4.00), 258 (3.46), 263 (3.49), 269
MeOH
max
(sh); 13C-NMR spectral data: see Table 1. The 13C-
1
and H-NMR spectral data of the sugar moiety were
in good agreement with those of 2.
were in good agreement with those of 2.
24
D
27
Compound 4. Amorphous powder; ‰aŠ
+30.98
Compound 8. Amorphous powder; ‰aŠ
+18.68
D
(MeOH; c 1.14); FABMS m/z: 899 [M + H]+, 921 [M
+ Na]+; 13C-NMR spectral data: see Table 1. The
13C- and 1H-NMR spectral data of the sugar moiety
(MeOH; c 1.17); FABMS m/z: 915 [M+H]+, 937
[M+Na]+; 13C NMR spectral data: see Table 1. The
13C and 1H NMR spectral data of the sugar moiety
were in good agreement with those of 2.
were in good agreement with those of 2.
Compound 9. Amorphous powder; ‰aŠ
24
D
24
D
Compound 5. Amorphous powder; ‰aŠ
+21.58
4.68
(MeOH; c 0.59); FABMS m/z: 941 [M + H]+, 963 [M
+ Na]+; 13C-NMR spectral data: see Table 1. The
13C- and 1H-NMR spectral data of the sugar moiety
(MeOH; c 0.61); FABMS m/z: 1020 [M + H]+; UV
MeOH
max
l
nm ꢀlog e): 218 (3.97), 258 (3.39), 263 (3.42), 270
(sh); 13C-NMR spectral data: see Table 1. The 13C-
1
were in good agreement with those of 2.
Compound 6. Amorphous powder; ‰aŠ
and H-NMR spectral data of the sugar moiety were
in good agreement with those of 2.
27
D
+12.18
(MeOH; c 0.52); FABMS m/z: 1051 [M + Na]+; UV
Compound 10. Amorphous powder; ‰aŠ
0.538
27
D
l
nm ꢀlog e): 217 (4.14), 223 (4.08), 278 (4.33);
(MeOH; c 1.23); FABMS m/z: 915 [M + H]+, 937 [M
+ Na]+; 13C-NMR spectral data: see Table 1. The
MeOH
max
13C-NMR spectral data: see Table 1. The 13C- and H-
1

492
T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
Table 2
13C-NMR spectral data of the sugar moiety (in pyridine-d5 solution at 358C)a
Carbon No.
1
2
13
16
19
28
31
33
34
Dig
96.4
38.7b
67.4c
83.4d
68.7
±
Dig
Dig
Dig
Cym
96.5
37.3b
78.1
83.4c
69.1
58.9
Dig
Cym
96.5
37.4b
78.2
83.4c
69.1
58.9
Dig
Cym
96.4
37.3b
78.1c
83.4d
69.0
59.0e
Cym
100.5
37.1b
78.0c
83.3d
69.0
58.9e
Dig
Cym
96.5
37.3b
78.0
83.4c
69.0
59.0d
Cym
100.5
37.3b
78.0
83.2c
69.0
58.9d
Dig
Cym
96.4
C-1'
C-2'
C-3'
C-4'
C-5'
OMe
96.4
38.7b
67.5c
83.5d
68.6e
±
96.4
38.7b
67.5c
83.5d
68.6e
±
96.4
38.7b
67.5c
83.5d
68.6
±
37.6b
78.1c
83.4d
69.0e
58.9f
Cym
100.3
37.4b
77.8c
83.2d
68.9e
58.8f
Ole
Dig
99.8
39.0b
67.6c
83.3d
68.7
±
Dig
Dig
Dig
C-10
C-20
C-30
C-40
C-50
OMe
99.9
39.1b
67.4c
83.3d
68.7e
±
99.8
39.1b
67.4c
83.2d
68.6e
±
99.8
39.1b
67.4c
83.2d
68.6
±
100.5
38.8b
67.5d
83.3c
68.7e
±
100.5
38.9b
67.5d
83.2c
68.6
±
Ole
101.6
37.0
81.4
76.2
73.0
57.0
Ole
±
Dig
Dig
Dig
Dig
Dig
C-1'0
C-2'0
C-3'0
C-4'0
C-5'0
OMe
99.9
38.5b
67.4c
83.1d
68.7e
±
99.8
38.6b
67.4c
83.1d
68.7e
±
99.8
38.6b
67.4c
83.1d
68.6
±
99.9
38.7
67.4d
83.1c
68.5e
±
99.9
38.7
67.5d
83.1c
68.6
±
100.6
38.9
67.5
83.2d
68.6
±
100.5
38.9
67.5
83.2c
68.5
±
102.0
37.2b
79.1
82.7d
71.7
57.3g
Ole
Ole
Ole
Ole
Ole
Ole
Ole
Ole
C-100
C-200
C-300
C-400
C-500
OMe
101.6
37.0
81.4
76.2
73.0
57.0
101.4
37.2
79.3
83.1
72.1
57.2
Glc
101.4
37.2
79.3
83.1
72.1
57.2
All
101.6
37.0b
81.4
76.2
73.0
57.0
101.4
37.2b
79.3
83.1c
72.1
57.2
Glc
101.6
37.0
81.4
76.2
73.0
57.0
101.4
37.1b
79.3
83.2c
72.1
57.2
Glc
100.5
37.1b
81.7
±
±
±
76.4
73.0
57.1g
±
±
C-1'00
C-2'00
C-3'00
C-4'00
C-5'00
C-6'00
C-6s
±
±
104.5
75.7
78.7
72.1
78.2
63.2
18.5 Â 2
18.7
18.8
102.1
72.8
73.2
69.5
75.7
63.4
18.5 Â 2
18.7
18.9
±
104.5
75.7
78.7
72.1
78.1
63.2
18.5 Â 2
18.6
18.8
±
104.5
75.8
78.7
72.1
78.2
63.2
18.2
18.5
18.6
18.7
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
18.5
18.6
18.7
18.5
18.6 Â 2
18.7
18.5 Â 2
18.6 Â 2
18.4
18.5
18.6 Â 2
18.5
18.6
18.7
18.8
a Dig: b-D-digitoxopyransyl, Cym: b-D-cymaropyranosyl, Ole: b-D-oleandropyranosyl, Glc: b-D-glucopyranosyl, All: b-D-allopyranosyl.
b±g Interchangeable within each column.
13C- and 1H-NMR spectral data of the sugar moiety
were in good agreement with those of 2.
13C-NMR spectral data of the aglycone moiety were in
good agreement with those of 5.
27
D
27
Compound 11. Amorphous powder; ‰aŠ
+6.58
Compound 14. Amorphous powder; ‰aŠ
0.838
D
(MeOH; c 0.73); FABMS m/z: 1083 [M + Na]+. The
(MeOH; c 1.03); FABMS m/z: 1141 [M + Na]+. The
13C- and 1H-NMR spectral data of the aglycone and
sugar moiety were in good agreement with those of 1
13C- and H-NMR spectral data of the aglycone moi-
1
ety and sugar moiety were in good agreement with
those of 3 and 13, respectively.
and 13, respectively.
27
D
21
D
Compound 12. Amorphous powder; ‰aŠ +28.38
Compound 15. Amorphous powder;‰aŠ
+1.98
(MeOH; c 1.12); FABMS m/z: 1083 [M + Na]+. The
13C- and 1H-NMR spectral data of the aglycone and
sugar moiety were in good agreement with those of 4
(MeOH; c 0.84); FABMS m/z: 1099 [M + Na]+. The
13C- and 1H-NMR spectral data of the aglycone and
sugar moiety were in good agreement with those of 10
and 13, respectively.
Compound 13. Amorphous powder; ‰aŠ
and 13, respectively.
Compound 16. Amorphous powder; ‰aŠ
27
D
27
D
13.88
+1.68
(MeOH; c 1.47); FABMS m/z: 1125 [M + Na]+; 13C-
and 1H-NMR spectral data: see Tables 2 and 3. The
(MeOH; c 0.41); FABMS m/z: 1099 [M + Na]+; 13C-
1
and H-NMR NMR spectral data: see Tables 2 and 3.

T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
493
Table 3
1H-NMR spectral data of the sugar moiety (in pyridine-d5 solution at 358C)a
Proton No.
1
2
13
Dig
Dig
Dig
H-1'
H-3'
H-4'
H-5'
H-6'
OMe'
5.46 (dd, 9.5, 2.0)
4.66 (br s)
3.50 (dd, 9.5, 3.0)
4.28 (dq, 9.5, 6.5)
1.43 (d, 6.5)
±
5.45 (dd, 9.5, 2.0)
4.63 (br s)
3.50 (dd, 9.5, 3.0)
4.28 (dq, 9.5, 6.5)
1.39 (d, 6.5)b
±
5.45 (dd, 9.5, 2.0)
4.63 (q, 3.0)
3.51 (dd, 9.5, 3.0)
4.28 (dq, 9.5, 6.5)
1.42 (d, 6.5)
±
Dig
Dig
Dig
H-10
H-30
H-40
H-50
H-60
OMe0
5.39 (dd, 9.5, 2.0)
4.63 (br s)
3.47 (dd, 9.5, 3.0)
4.30 (dq, 9.5, 6.5)
1.40 (d, 6.5)
±
5.36 (dd, 9.5, 2.0)
4.63 (br s)
3.44 (Ã)e
5.36 (dd, 9.5, 2.0)
4.62 (q, 3.0)
3.44 (dd, 9.5, 3.0)
4.25 (dq, 9.5, 6.5)
1.33 (d, 6.5)
±
4.25 (dq, 9.5, 6.5)
1.33 (d, 6.5)
±
Ole
4.79 (dd, 9.5, 2.0)
3.45 (Ã)e
Dig
Dig
H-1'0
H-3'0
H-4'0
H-5'0
H-6'0
OMe'0
5.36 (dd, 9.5, 2.0)
4.63 (br s)
3.47 (Ã)e
5.36 (dd, 9.5, 2.0)
4.58 (q, 3.0)
3.41 (dd, 9.5, 3.0)
4.26 (dq, 9.5, 6.5)
1.35 (d, 6.5)
±
3.43 (Ã)e
3.57 (dq, 8.5, 6.0)
1.49 (d, 6.0)
3.46 (s)
4.28 (dq, 9.5, 6.5)
1.42 (d, 6.5)b
±
Ole
4.79 (dd, 9.5, 2,0)
3.44 (Ã)e
Ole
4.72 (dd, 9.5, 2.0)
3.63 (Ã)e
H-100
H-300
H-400
H-500
H-600
OMe00
±
±
±
±
±
±
3.41 (Ã)e
3.63 (Ã)e
3.57 (dq, 8.5, 6.0)
1.49 (d, 6.0)
3.46 (s)
3.64 (Ã)e
1.64 (d, 6.0)
3.52 (s)
Glc
H-1'00
H-2'00
H-3'00
H-4'00
H-5'00
H-6'00
±
±
±
±
±
±
±
±
±
±
±
±
5.10 (d, 8.0)
3.98 (t, 8.0)
4.20 (t, 8.0)b
4.17 (t, 8.0)b
3.93 (m)
4.33 (dd, 11.5, 5.5)
4.51 (dd, 11.5, 2.5)
Proton No.
16
19
28
Dig
5.45 (dd, 9.5, 2.0)
4.62 (br s)
Cym
Cym
H-1'
H-3'
H-4'
H-5'
H-6'
OMe'
5.26 (dd, 9.5, 2.0)
4.09 (q, 3.0)
3.52 (dd, 9.5, 3.0)
4.21 (dq, 9.5, 6.5)
1.38 (d, 6.5)
3.62 (s)
5.28 (dd, 9.5, 2.0)
4.09 (q, 3.0)
3.52 (Ã)e
3.49 (dd, 9.5, 3.0)
4.26 (dq, 9.5, 6.5)
1.39 (d, 6.5)
±
4.21 (Ã)e
1.38 (d, 6.5)b
3.62 (s)
Dig
Dig
5.35 (dd, 9.5, 2.0)
4.62 (br s)
Dig
5.32 (dd, 9.5, 2.0)
4.63 (br s )
H-10
H-30
H-40
H-50
H-60
OMe0
5.32 (dd, 9.5, 2.0)
4.63 (q, 3.0)
3.47 (dd, 9.5, 3.0)
4.24 (dq, 9.5, 6.5)
1.38 (d, 6.5)b
±
3.43 (dd, 9.5, 3.0)
4.24 (dq, 9.5, 6.5)
1.32 (d, 6.5)
±
3.48 (dd, 9.5, 3.0)
4.24 (dq, 9.5, 6.5)
1.38 (d, 6.5)
±
Dig
5.35 (dd, 9.5, 2.0)
4.62 (br s)
Dig
5.38 (dd, 9.5, 2.0)
4.63 (br s )
Dig
H-1'0
H-3'0
H-4'0
H-5'0
5.36 (dd, 9.5, 2.0)
4.59 (q, 3.0)
3.42 (dd, 9.5, 3.0)
4.28 (dq, 9.5, 6.5)
(continued on next page)
3.40 (dd, 9.5, 3.0)
4.26 (dq, 9.5, 6.5)
3.48 (dd, 9.5, 3.0)
4.30 (dq, 9.5, 6.0)

494
T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
Table 3 (continued )
Proton No.
16
19
28
H-6'0
OMe'0
1.35 (d, 6.5)
1.41 (d, 6.0))
±
Ole
1.36 (d, 6.5)b
±
Ole
±
Ole
H-100
H-300
H-400
H-500
H-600
OMe00
4.71 (dd, 9.5, 2.0)
4.80 (dd, 9.5, 2.0)
3.45 (Ã)e
4.73 (dd, 9.5, 2.0)
3.62 (Ã)e
3.43 (Ã)e
3.62 (Ã)e
3.60 (Ã)e
3.57 (dq, 8.5, 6.0)
1.50 (d, 6.0)
3.46 (s)
3.65 (Ã)e
1.60 (d, 6.0)
3.53 (s)
All
1.64 (d, 6.0)
3.52 (s)
Glc.
H-1'00
H-2'00
H-3'00
H-4'00
H-5'00
H-6'00
5.50 (d, 8.0)
3.93 (br s)
4.69 (t, 2.5)
4.17 (br d, 9.5)
4.43 (Ã)e
±
±
±
±
±
±
±
5.10 (d, 8.0)
3.99 (t, 8.0)
4.20 (Ã)e
4.17 (t, 8.5)
3.93 (m)
4.32 (Ã)e
4.33 (dd, 12.0, 5.5)
4.52 (dd, 12.0, 2.0)
4.47 (dd, 11.5, 3.0)
Proton No.
31
33
34
Cym
Cym
Cym
5.26 (dd, 9.5, 2.0)
4.08 (q, 3.0)
H-1'
H-3'
H-4'
H-5'
H-6'
OMe'
5.29 (dd, 9.5, 2.0)
4.07 (q, 3.0)
3.49 (dd, 9.5, 3.0)
4.22 (dq, 9.5, 6.5)
1.38 (d, 6.5)
3.63 (s)
5.25 (dd, 9.5, 2.0)
4.06 (q, 3.0)
3.48 (dd, 9.5, 3.0)
4.19 (Ã)e
4.17 (dq, 9.5, 6.5)
1.36 (d, 6.5)b
3.62 (s)c
1.36 (d, 6.5)
3.62 (s)
Cym
Cym
Cym
5.11 (dd, 9.5, 2.0)
4.03 (q, 3.0)
H-10
H-30
H-40
H-50
H-60
OMe0
5.12 (dd, 9.5, 2.0)
4.08 (q, 3.0)
3.48 (dd, 9.5, 3.0)
4.16 (dq, 9.5, 6.5)
1.33 (d, 6.5)
3.62 (s)
5.11 (Ã)e
4.07 (q, 3.0)
3.47 (dd, 9.5, 3.0)
4.15 (dq, 9.5, 6.5)
1.33 (d, 6.5)
3.61 (s)
4.19 (dq, 9.5, 6.5)
1.39 (d, 6.5)b
3.59 (s)c
Dig
5.32 (dd, 9.5, 2.0)
4.63 (br s)
Dig
Ole
4.71 (dd, 9.5, 2.0)
H-1'0
H-3'0
H-4'0
H-5'0
H-6'0
OMe'0
5.30 (dd, 9.5, 2.0)
4.59 (q, 3.0)
3.43 (dd, 9.5, 3.0)
4.27 (dq, 9.5, 6.5)
1.41 (d, 6,5)
±
3.51 (dd, 9.5, 3.0)
4.29 (dq, 9.5, 6.5)
1.46 (d, 6.5)
±
1.48 (d, 6.0)
3.52 (s)d
Ole
Ole
4.82 (dd, 9.5, 2.0)
3.46 (Ã)e
Ole
4.74 (dd, 9.5, 2.0)
3.63 (Ã)e
H-100
H-300
H-400
H-500
H-600
OMe00
4.99 (dd, 9.5, 2.0)
3.42 (Ã)e
3.65 (Ã)e
3.59 (dq, 8.5, 6.0)
1.51 (d, 6,0)
3.46 (s)
3.63 (Ã)e
1.65 (d, 6.0)
3.52 (s)
Glc
1.59 (d, 6.0)
3.48 (s)d
H-1'00
H-2'00
H-3'00
H-4'00
H-5'00
H-6'00
±
±
±
±
±
±
±
5.10 (d, 8.0)
3.99 (t, 8.0)
4.19 (Ã)e
±
±
±
±
±
±
±
4.17 (Ã)e
3.93 (m)
4.33 (dd, 11.5, 4.5)
4.52 (dd, 11.5, 2.5)
a
1
Assignments were done based on the 2D-NMR ꢀ H±1H COSY, HMQC and HMBC) spectra.
b±d Interchangeable within each column.
e (Ã) Overlapping with other signals.

T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
495
Table 4
13C- and 1H-NMR spectral data of compound 2 (in CDCl3 solution at 358C)a
Aglycone moiety
Sugar moiety
C-1
C-2
38.8
28.8
77.8
38.8
141.1
117.3
34.1
74.5
43.7
37.2b
24.2
72.5
57.8
88.2
32.6
32.2
91.7
9.2
Dig-1'
Dig-2'
Dig-3'
Dig-4'
Dig-5'
Dig-6'
95.9
4.92 (dd, 9.5, 2.0)
37.1b
66.4c
82.2d
68.1e
18.2
C-3
C-4
3.57 (m)
4.24 (Ã)f
3.22 (dd, 9.5, 3.0)
3.78 (dq, 9.5, 6.5)
1.22 (d, 6.5)
C-5
C-6
±
5.34 (br s)
C-7
C-8
±
±
Dig-10
Dig-20
Dig-30
Dig-40
Dig-50
Dig-60
98.3
36.7
66.5c
82.4d
68.2e
18.2
4.88 (dd, 9.5, 2.0)
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
4.24 (Ã)f
3.20 (dd, 9.5, 3.0)
3.81 (dq, 9.5, 6.5)
1.23 (d, 6.5)
4.51 (dd, 10.5, 6.0)
±
±
Dig-1'0
Dig-2'0
Dig-3'0
Dig-4'0
Dig-5'0
Dig-6'0
98.3
36.7
66.5c
82.5d
68.3e
18.2
4.90 (dd, 9.5, 2.0)
±
4.24 (Ã)f
1.42 (s)
1.12 (s)
±
3.20 (dd, 9.5, 3.0)
3.84 (dq, 9.5, 6.5)
1.24 (d, 6.5)
18.7
209.3
27.7
2.24 (s)
Ole-100
Ole-200
Ole-300
Ole-400
Ole-500
Ole-600
Ole-OMe00
100.4
35.3
80.4
75.3
71.9
17.9
56.4
4.55 (dd, 9.5, 2.0)
Ac-1'
Ac-2'
169.8
20.7
±
1.94 (s)
3.18 (Ã)f
3.12 (t, 8.5)
3.33 (dq, 8.5, 6.0)
1.32 (d, 6.0)
3.40 (s)
a Dig: b-D-digitoxopyranosyl, Ole: b-D-oleandropyranosyl.
b±e Interchangeable within each coloumn.
f (Ã): Overlapping with other signals.
The 13C-NMR spectral data of the aglycone moiety
were in good agreement with those of 10.
(MeOH; c 0.33); FABMS m/z: 1019 [M + H]+; UV
MeOH
max
l
nm ꢀlog e): 219 (4.00), 263 (3.49). The 13C- and
27
D
1H-NMR spectral data of the aglycone and sugar moi-
ety were in good agreement with those of 7 and 19, re-
Compound 17. Amorphous powder; ‰aŠ
+8.98
(MeOH; c 0.97); FABMS m/z: 913 [M + H]+, 935 [M
+ Na]+. The 13C- and H-NMR spectral data of the
1
spectively.
27
D
aglycone and sugar moiety were in good agreement
with those of 3 and 19, respectively.
Compound 22. Amorphous powder; ‰aŠ
+2.28
(MeOH; c 0.74); FABMS m/z: 971 [M + H]+, 993 [M
20
D
+ Na]+. The 13C- and H-NMR spectral data of the
1
Compound 18. Amorphous powder; ‰aŠ +35.58
(CHCl3; c 0.78); FABMS m/z: 913 [M + H]+, 935 [M
aglycone and sugar moiety were in good agreement
with those of 1 and 19, respectively.
+ Na]+. The 13C- and H-NMR spectral data of the
1
aglycone and sugar moiety were in good agreement
with those of 4 and 19, respectively.
27
D
Compound 23. Amorphous powder; ‰aŠ
+3.78
(MeOH; c 0.79); FABMS m/z: 929 [M + H]+, 951 [M
23
D
Compound 19. Amorphous powder; ‰aŠ
16.78
+ Na]+. The 13C- and H-NMR spectral data of the
1
(MeOH; c 0.66); FABMS m/z: 955 [M + H]+, 977 [M
aglycone and sugar moiety were in good agreement
with those of 10 and 19, respectively.
27
+ Na]+; 13C- and H-NMR spectral data: see Tables
1
2 and 3. The 13C-NMR spectral data of the aglycone
moiety were in good agreement with those of 5.
Compound 24. Amorphous powder; ‰aŠ +25.48
D
(MeOH; c 0.40); FABMS m/z: 929 [M + H]+, 951 [M
+ Na]+; 13C-NMR spectral data: see Table 1. The
13C- and 1H-NMR spectral data of the sugar moiety
27
D
Compound 20. Amorphous powder; ‰aŠ +14.48
(MeOH; c 0.89); FABMS m/z: 1065 [M + Na]+; UV
MeOH
max
l
nm ꢀlog e): 217 (4.19), 222 (4.12), 278 (4.39). The
13C- and 1H-NMR spectral data of the aglycone and
sugar moiety were in good agreement with those of 6
and 19, respectively.
were in good agreement with those of 19.
27
D
Compound 25. Amorphous powder; ‰aŠ +18.78
(MeOH; c 0.78); FABMS m/z: 1097 [M + Na]+. The
13C- and 1H-NMR spectral data of the aglycone and
21
D
Compound 21. Amorphous powder; ‰aŠ
14.48

496
T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
sugar moiety were in good agreement with those of 3
and 28, respectively.
and extracted with EtOAc. The EtOAc layer was con-
centrated to dryness. Puri®cation of the residue by
HPLC (YMC-ODS, 30, 35, 52.5% MeOH in water
and 25, 27.5% MeCN in water) a€orded lineolon (35,
12 mg), isolineolon (36, 2 mg), 12-O-acetyllineolon (37,
2 mg), deacylmetaplexigenin (38, 4 mg), metaplexigenin
(39, 5 mg) and 15b-hydroxylineolon (40, 28 mg) and
21
Compound 26. Amorphous powder; ‰aŠ +32.18
D
(MeOH; c 0.90); FABMS m/z: 1097 [M + Na]+. The
13C- and 1H-NMR spectral data of the aglycone and
sugar moiety were in good agreement with those of 4
and 28, respectively.
27
D
Compound 27. Amorphous powder; ‰aŠ
10.08
15b-hydroxyisolineolon (41, 5 mg).
(MeOH; c 1.01); FABMS m/z: 1139 [M + Na]+. The
13C- and 1H-NMR spectral data of the aglycone and
sugar moiety were in good agreement with those of 5
Compound 37. ‰aŠ
70.28 (MeOH; c 0.26); FABMS
21
D
m/z: 407 [M + H]+, 429 [M + Na]+; 13C-NMR spec-
tral data (pyridine-d5 at 358C): d 209.7 (C-20), 167.0
(CÃOCH3), 140.5 (C-5), 118.5 (C-6), 87.5, (C-14),
74.7 (C-8), 73.3 (C-12), 71.6 (C-3), 60.5 (C-17),
55.7 (C-13), 44.9 (C-9), 43.3 (C-4), 39.3 (C-1), 37.5
(C-10), 35.2, 34.2 (C-7, 15), 32.3 (C-21), 32.0 (C-2),
24.9 (C-11), 21.8 (C-16), 20.8 (COCÃH3), 18.4 (C-19),
15.7 (C-18). 1H-NMR spectral data (pyridine-d5 at
358C): d 5.32 (br s, H-6), 5.05 (dd, 12.0, 4.0, H-12),
3.87 (m, H-3), 3.49 (t, 9.5, H-17), 2.26 (s, H-21), 2.05
(s, COCHÃ3), 1.90 (s, H-18), 1.72 (dd, 12.5, 3.5, H-9),
and 28, respectively.
Compound 28. Amorphous powder; ‰aŠ
27
D
12.18
(MeOH; c 0.45); FABMS m/z: 1180 [M + H]+, 1202
[M + Na]+; UV l
nm ꢀlog e): 219 (4.07), 259
MeOH
max
(3.45), 263 (3.49), 270 (sh); 13C- and H-NMR spectral
data: see Tables 2 and 3. The 13C-NMR spectral data
of the aglycone moiety were in good agreement with
1
those of 7.
27
D
Compound 29. Amorphous powder; ‰aŠ
+1.38
(MeOH; c 0.65); FABMS m/z: 1155 [M + Na]+. The
13C- and 1H-NMR spectral data of the aglycone and
sugar moiety were in good agreement with those of 1
1.40 (s, H-19).
Compound 40. ‰aŠ
21
D
3.48 (MeOH; c 0.85); FABMS
m/z: 381 [M + H]+; 13C-NMR spectral data (pyri-
dine-d5 at 358C): d 209.8 (C-20), 139.8 (C-5), 119.1 (C-
6), 86.4 (C-14), 74.8 (C-8), 71.7 Â 2 (C-3, 15), 68.7 (C-
12), 59.4 (C-17), 58.2 (C-13), 45.3 (C-9), 43.5 (C-4),
39.3 (C-1), 37.6 (C-10), 35.4 (C-7), 34.5 (C-16), 32.2
(C-2), 32.1 (C-21), 29.3 (C-11), 18.8 (C-19), 15.2 (C-
18). 1H-NMR spectral data (pyridine-d5 at 358C): d
5.45 (br s, H-6), 5.07 (s, 14-OHÃ), 4.31 (s, 8-OHÃ), 4.54
(m, H-15), 4.03 (dd, 12.0, 4.0, H-12), 3.86 (m, H-3),
3.53 (t, 9.5, H-17), 3.33 (dt, 14.5, 10.0, H-16), 2.42 (s,
H-21), 2.03 (s, H-18), 1.67 (dd, 13.5, 2.5, H-9), 1.48 (s,
and 28, respectively.
Compound 30. Amorphous powder; ‰aŠ +14.28
27
D
(MeOH; c 0.46); FABMS m/z: 927 [M + H]+, 949 [M
+ Na]+. The 13C- and H-NMR spectral data of the
1
aglycone and sugar moiety were in good agreement
with those of 3 and 31, respectively.
27
D
Compound 31. Amorphous powder; ‰aŠ +46.28
(MeOH; c 0.50); FABMS m/z: 927 [M + H]+, 949 [M
+ Na]+; 13C- and H-NMR spectral data: see Tables
1
2 and 3. The 13C-NMR spectral data of the aglycone
moiety were in good agreement with those of 4.
H-19).
27
D
21
D
Compound 32. Amorphous powder; ‰aŠ
2.78
Compound 41. ‰aŠ
+55.98 (MeOH; c 0.47);
(MeOH; c 0.17). FABMS m/z: 991 [M + Na]+. The
FABMS m/z: 381 [M + H]+, 403 [M + Na]+; 13C-
NMR spectral data (pyridine-d5 at 358C): d 215.3 (C-
20), 139.3 (C-5), 119.5 (C-6), 84.7 (C-14), 75.0, 74.9
(C-8, 15), 73.5 (C-12), 71.7 (C-3), 56.3 Â 2 (C-13, 17),
45.9 (C-9), 43.5 (C-4), 39.4 (C-1), 37.7 (C-10), 36.0 (C-
16), 35.1 (C-7), 32.3 (C-2), 31.9 (C-21), 28.5 (C-11),
13C- and H-NMR spectra of the aglycone and sugar
1
moiety were in good agreement with those of 5 and
31, respectively.
21
D
Compound 33. Amorphous powder; ‰aŠ
+7.98
(MeOH; c 0.63); FABMS m/z: 1127 [M + Na]+; 13C-
and 1H-NMR spectral data: see Tables 2 and 3. The
13C-NMR spectral data of the aglycone moiety were in
1
18.9 (C-19), 12.5 (C-18). H-NMR spectral data (pyri-
dine-d5 at 358C): d 5.47 (br s, H-6), 5.30 (s, 14-OHÃ),
4.58 (br s, H-15), 4.23 (s, 8-OHÃ), 3.89 (m, H-3), 3.83
(dd, 12.0, 4.0, H-12), 3.78 (dd, 9.5, 5.0, H-17), 2.61 (dt,
14.0, 9.5, H-16), 2.32 (overlapped, H-16), 2.30 (s, H-
21), 1.78 (dd, 13.5, 2.5, H-9), 1.66 (s, H-18), 1.50 (s, H-
19).
The H2O layer was neutralized with Amberlite IRA-
60E and the eluate was concentrated to dryness. The
residue was chromatographed on a silica gel column
with CHCl3±MeOH±H2O (7:1:1.2 bottom layer) sys-
tem to obtain cymarose (13 mg), oleandrose (37 mg),
digitoxose (57 mg).
good agreement with those of 10.
27
D
Compound 34. Amorphous powder; ‰aŠ
2.28
(MeOH; c 0.80); FABMS m/z: 979 [M + Na]+; 13C-
and 1H-NMR spectral data: see Tables 2 and 3. The
13C-NMR spectral data of the aglycone moiety were in
good agreement with those of 10.
3.3. Acid hydrolysis of a mixture of pregnane glycosides
A mixture of pregnane glycosides (510 mg) was
heated at 608C for 5 h with dioxane (8 ml) and 0.2 N
H2SO4 (2 ml) to yield the aglycones and sugars. After
hydrolysis, this reaction mixture was diluted with H2O
All these monosaccharides were believed to be of D-
form based on their optical rotation values (Tsuka-

T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
497
moto et al., 1986; Nakagawa et al., 1983; Abe et al.,
1994).
ness. Boric acid was removed by co-distillation with
MeOH, and the residue was acetylated overnight with
acetic anhydride and pyridine (4 drops each) at room
temperature. After evaporation of the reagents under a
stream of air, cymaritol acetate, oleandritol acetate,
digitoxitol acetate, glucitol acetate and allitol acetate
were detected by GC (conditions: column, Supelco SP-
2380TM capillary column 0.25 mm  30 m, carrier gas
N2, column temperature 2008C;Rt, cymaritol acetate
7.5 min, oleandritol acetate 8.5 min, digitoxitol acetate
10.9 min, column temperature 2508C;Rt, allitol acetate
9.4 min, glucitol acetate 12.4 min).
21
D
D-Cymarose: ‰aŠ +51.78 (c = 1.29, 24 h after
21
dissolution in H2O).
D-Oleandrose: ‰aŠ
11.78 (c = 1.87, 24 h after
D
dissolution in H2O).
D-Digitoxose: ‰aŠ +45.08 (c = 1.04, 24 h after
21
D
dissolution in H2O).
3.4. Acid hydrolysis of a mixture of pregnane glycosides
to determine the con®guration of glucose
A mixture of pregnane glycosides (ca. 10 mg) was
heated at 958C for 1.5 h with dioxane and 0.05 N HCl
(10 drops each). After hydrolysis, the reaction mixture
was passed through an Amberlite IRA-60E column,
and the eluate was evaporated under reduced pressure.
The residue was partitioned between H2O and EtOAc,
and the H2O layer was concentrated to dryness. This
residue was stirred with D-cysteine methyl ester hydro-
chloride (3 mg) (Hara, Okabe & Mihashi, 1987) in pyr-
idine (25 ml) at 608C for 1.5 h. Subsequently,
hexamethyldisilazane (10 ml) and trimethlsilylchloride
(10 ml) were added to the solution, and stirring was
continued at 608C for another 30 min. The precipitate
was removed with centrifugation, with the supernatant
next subjected to GC analysis. GC conditions: column,
GL Capillary Column TC-1 (GL Sciences) 0.32 mm Â
30 m, carrier gas N2, column temperature 2108C;Rt, D-
glucose 18.8 min, L-glucose 17.5 min. D-Glucose was
detected from the mixture of pregnane glycosides.
3.6. Acid hydrolysis of compound 16
Acid hydrolysis of 16 (3 mg) with 0.05 N HCl and a
reaction of the product following hydrolysis with D-
cysteine methyl ester hydrochloride, hexamethyldisila-
zane and trimethlsilylchloride was carried out as
described previously. GC analysis was used with the
same conditions as for the detection of D-glucose. Rt,
D-allose 19.0 min, L-allose 17.9 min. The Rt for L-
allose was obtained from its enantiomer (D-allose + L-
cysteine). D-Allose was detected from 16.
3.7. Enzymatic hydrolysis of compounds 11±15, 25±29
and 33
Compounds 11±15, 25±29 and 33 (ca. 2 mg) were
dissolved in H2O (0.7 ml), and cellulase (EC 3.2.1.4
from Penicillium funiculosum, 6.1 units/mg solid,
Sigma) (ca. 20±30 mg) was added. The mixture was
stirred at 408C for 1 day. After hydrolysis, the reaction
mixture was diluted with H2O and extracted with
EtOAc. The EtOAc extract of each compound except
for 33 contained 3, 4, 5, 2, 10, 17, 18, 19, 21 and 22,
whose structures were con®rmed by comparison of
3.5. Acid hydrolysis of compounds 1±34
Solutions of compounds 1±34 (ca. 0.5 mg) in diox-
ane and 0.05 N HCl (2 drops each) were heated at
958C for 1.5 h. After hydrolysis, this solution was
passed through an Amberlite IRA-60E column and
concentrated to dryness. The residues from 1±34 were
partitioned between EtOAc and H2O, and the EtOAc
extract was analyzed by HPLC to identify the aglycone
via comparison with authentic samples (conditions:
column, YMC-ODS 4.6 mm  25 cm; ¯ow rate, 1.0
ml/min, 30% MeOH in water;Rt, 15b-hydroxylineolon
12.0 min, 15b-hydroxyisolineolon 12.8 min, 35%
MeOH in water;Rt, lineolon 14.6 min, deacylmetaplexi-
genin 12.8 min, 42.5% MeOH in water;Rt, isolineolon
12.8 min, 50% MeOH in water;Rt, 12-O-acetyllineolon
14.4 min, 12-O-nicotinolylineolon 15.6 min, rostrata-
mine 17.2 min, 52.5% MeOH in water;Rt, metaplexi-
genin 15.6 min, 65% MeOH in water;Rt, ikemagenin
21.0 min).
1
their H-NMR spectra and HPLC retention times with
1
those of authentic samples. In the H-NMR spectrum
of the hydrolysis product of 33, the signals of the
sugar moiety were consistent with those of 31 (HPLC
conditions: column, YMC-ODS 4.6 mm  25 cm; ¯ow
rate, 1.0 ml/min, 60% MeOH in water: Rt, 10 14.2
min, 65% MeOH in water: Rt, 3 13.6 min, 70%
MeOH in water: Rt, 2 17.2 min, 4 11.2 min, 5 13.8
min, 17 11.4 min, 75% MeOH in water: Rt, 18 10.0
min, 19 12.4 min, 21 10.8 min, 22 16.4 min).
Acknowledgements
We thank the sta€ of the Central Analytical Labora-
tory of this university for measurements of FABMS,
and wish to acknowledge the technical support of Dr.
Toshio Miyase in determining the con®guration of glu-
cose and allose.
Subsequently, the H2O layer was reduced with
NaBH4 (ca. 1 mg) for 1 h at room temperature. The
reaction mixture was passed through an Amberlite IR-
120B column and the eluate was concentrated to dry-

498
T. Warashina, T. Noro / Phytochemistry 53 (2000) 485±498
G, ®ve additional new oxypregnane-oligoglycosides from the root
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