Polyhydroxylated spirostanol saponins from the tubers of Dioscorea polygonoides

Article abstract of DOI:10.1021/np0580356

Three new polyhydroxylated spirostanol saponins (1-3) were isolated from the tubers of Dioscorea polygonoides. The structures of these new compounds were determined on the basis of extensive spectroscopic analysis and the results of acid or enzymatic hydrolysis as (23S,24R,25S)-23,24-dihydroxyspirost-5-en- 3β-yl O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (1), (23S,25R)-12α,17α,23-trihydroxyspirost-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (2), and (23S,25R)-14α,17α,23-trihydroxyspirost-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (3), respectively.

Full text of DOI:10.1021/np0580356

1116
J. Nat. Prod. 2005, 68, 1116-1120
Polyhydroxylated Spirostanol Saponins from the Tubers of Dioscorea
polygonoides
Jaime Nin˜o Osorio,† Oscar M. Mosquera Martinez,† Yaned M. Correa Navarro,† Kazuki Watanabe,‡
Hiroshi Sakagami,§ and Yoshihiro Mimaki*,‡
Grupo de Biotecnologı´a-Productos Naturales, Escuela de Tecnologı´a Quı´mica, Universidad Tecnolo´gica de Pereira, La Julita,
A. A. 97 Pereira, Colombia, Laboratory of Medicinal Pharmacognosy, Tokyo University of Pharmacy and Life Science, School
of Pharmacy, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan, and Department of Dental Pharmacology, Meikai
University School of Dentistry, 1-1, Keyaki-dai, Sakado, Saitama 350-0283, Japan
Received March 7, 2005
Three new polyhydroxylated spirostanol saponins (1-3) were isolated from the tubers of Dioscorea
polygonoides. The structures of these new compounds were determined on the basis of extensive
spectroscopic analysis and the results of acid or enzymatic hydrolysis as (23S,24R,25S)-23,24-dihydroxy-
spirost-5-en-3â-yl O-R-L-rhamnopyranosyl-(1f2)-â-D-glucopyranoside (1), (23S,25R)-12R,17R,23-trihy-
droxyspirost-5-en-3â-yl O-R-L-rhamnopyranosyl-(1f2)-â-D-glucopyranoside (2), and (23S,25R)-14R,17R,23-
trihydroxyspirost-5-en-3â-yl O-R-L-rhamnopyranosyl-(1f2)-â-D-glucopyranoside (3), respectively.
Some Dioscorea species are useful not only as sources
for the preparation of steroidal saponins of commercial
value, or as sources of steroidal hormones, but also as folk
medicines.1 Dioscorea polygonoides Humb. et Bonpl.
(Dioscoreaceae) is distributed from Mexico to Brazil includ-
ing Colombia. A phytochemical investigation of the tubers
of D. polygonoides was carried out, with particular atten-
tion paid to the steroidal saponin constituents of the tubers,
and has resulted in the isolation of three new polyhydroxy-
lated spirostanol saponins (1-3). This paper deals with the
structural determination of 1-3 on the basis of extensive
spectroscopic analysis and the results of acid or enzymatic
hydrolysis.
strong IR absorptions at 3379 and 1044 cm-1. The 1H NMR
spectrum of 1 in pyridine-d5 displayed the following
representative signals: four steroid methyl protons at δ
1.18 (d, J ) 7.0 Hz), 1.09 (d, J ) 6.5 Hz), 1.03 (s), and 0.98
(s); an olefinic proton at δ 5.28 (br d, J ) 4.9 Hz); two
anomeric protons at δ 6.33 (d, J ) 0.6 Hz) and 5.00 (d, J )
7.7 Hz); and the methyl group of a 6-deoxyhexopyranose
unit at δ 1.75 (d, J ) 6.2 Hz). Enzymatic hydrolysis of 1
using naringinase failed to cleave the sugar moiety of 1
due to the insoluble nature of this glycoside in aqueous
solution. Acid hydrolysis of 1 with 1.0 M HCl in dioxane-
H2O (1:1) gave D-glucose and L-rhamnose as the carbohy-
drate components, while the labile aglycon decomposed
under acid conditions. The above data, along with two
anomeric carbon signals observed at δ 102.0 and 100.3 and
one distinctive quaternary carbon resonance appearance
at δ 113.2, led to the realization that 1 is a spirostanol
1
diglycoside.2 Comparison of the H and 13C NMR assign-
ments of the aglycon moiety, which were established by
analysis of the 1H-1H COSY, HMQC, and HMBC spectra,
with data of (25R)-spirost-5-en-3â-ol (diosgenin) 3-O-gly-
coside, isolated from several plants in the Liliaceae,3-5
revealed that the structure of the ring A-E portion of the
molecule (C-1-C-21) was identical to that of this reference
compound, including the orientations of the C-3 oxygen
atom (3â-equatorial) and Me-21 group (20R) and the ring
junctions (B/C trans, C/D trans, D/E cis). However, signifi-
cant differences were recognized in the signals from the
ring F portion (C-22-C-27). The H-1H COSY spectrum
1
A MeOH extract of the tubers of D. polygonoides was
partitioned between n-BuOH saturated with H2O and H2O.
The n-BuOH-soluble phase was subjected to column chro-
matography on silica gel and octadecylsilanized (ODS)
silica gel, as well as preparative HPLC, yielding 1 (12.6
mg), 2 (74.4 mg), and 3 (34.1 mg).
Compound 1, isolated as an amorphous solid, showed an
accurate [M + H]+ ion at m/z 755.4213 in the positive-ion
HRESIMS, corresponding to the empirical molecular for-
mula C39H62O14. The glycosidic nature of 1 was shown by
was carefully inspected to assign the structure of the ring
F residue, with the three-proton doublet at δ 1.09 (J ) 6.5
Hz), attributable to Me-27, being used as the starting point
for analysis. The Me-27 protons showed a spin-coupling
correlation with the broad multiplet centered at δ 2.04,
which was unambiguously assigned to H-25 and exhibited
correlations with a pair of oxymethylene protons at δ 3.66
and 3.64 (H2-26) and the oxymethine proton at δ 3.97 (H-
24). The oxymethine proton, in turn, displayed a correlation
with the terminal oxymethine proton at δ 3.88 (H-23).
These subsequent correlations led the ring F fragment of
1 to be proposed as -C(23)H(O-)-C(24)H(O-)-C(25)H-
(C(27)H3)-C(26)H2-O-. Thus, the presence of oxygen atoms
at C-23 and C-24 was evident. The proton spin-coupling
* To whom correspondence should be addressed. Tel: +81-426-76-4577.
Fax: +81-426-76-4579. E-mail: mimakiy@ps.toyaku.ac.jp.
† Universidad Tecnolo´gica de Pereira.
‡ Tokyo University of Pharmacy and Life Science.
§ Meikai University School of Dentistry.
10.1021/np0580356 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/06/2005

Notes
Journal of Natural Products, 2005, Vol. 68, No. 7 1117
constant between H-23 and H-24 (J ) 9.4 Hz), between
H-24 and H-25 (J ) 9.4 Hz), and between H-25 and
H-26axial (J ) 11.3 Hz), along with NOE correlations from
H-23 to H-20, Me-21, and H-25 and from H-26axial to H-16
and H-24 in the PHNOSY spectrum of 1, were consistent
with the 22R, 23S, 24R, and 25S configurations. Treatment
of 1 with Ac2O in pyridine gave the corresponding octaac-
Me-18 to H-8, H-15â, and H-20, Me-19 to H-8 and H-11axial,
and H-23 to H-20, Me-21, and H-25, along with the proton
spin-coupling constants of H-12 (br s), H-23 (dd, J ) 11.2,
4.6 Hz), and H-26axial (dd, J ) 11.2, 11.2 Hz), confirmed
the B/C trans, C/D trans, and D/E cis ring junctions and
the 12R, 17R, 20R, 22R, 23S, and 25R configurations. The
diglycoside O-R-L-rhamnosyl-(1f2)-â-D-glucosyl group was
ascertained to be linked to C-3 of the aglycon by comparison
of the 1H and 13C NMR spectra of 2 with those of 1 and by
analysis of the HMBC spectrum of 2. Thus, the structure
of 2 was assigned as (23S,25R)-12R,17R,23-trihydroxy-
spirost-5-en-3â-yl O-R-L-rhamnopyranosyl-(1f2)-â-D-glu-
copyranoside.
1
etate (1a). When the H NMR spectrum of 1a was com-
pared with that of 1, the H-23 and H-24 protons were
moved downfield by 1.48 and 1.54 ppm and were observed
at δ 5.36 and 5.51, respectively, whereas the chemical shift
of H-3 was almost unaffected. This indicated that C-23 and
C-24 have a free hydroxy group and that C-3 is glycosy-
1
lated. Analysis of the H and 13C NMR spectra and the
Compound 3 was analyzed for C39H62O15 as determined
by the positive-ion HRESIMS (m/z 793.3992 [M + Na]+).
The 1H NMR spectrum of 3 showed signals for four steroid
methyls at δ 1.34 (d, J ) 7.3 Hz), 1.31 (s), 1.04 (s), and
0.68 (d, J ) 5.9 Hz) and two anomeric protons at δ 6.33 (d,
J ) 1.0 Hz) and 4.98 (d, J ) 7.7 Hz). Enzymatic hydrolysis
of 3 with naringinase furnished a new steroidal sapogenin
(3a: C27H42O6) and D-glucose and L-rhamnose. The molec-
ular formula of 3a was the same as that of 2a, and the 1H
NMR spectrum was essentially analogous to that of 2a,
showing signals for four exchangeable protons at δ 5.28,
4.94, 4.59, and 4.51, together with signals for four steroid
methyls. These data implied that 3a is an isomer of 2a with
regard to the linkage positions of the hydroxy groups to
the aglycon. The structure of 3a was determined by the
following spectroscopic data. The multiplet proton signal
centered at δ 3.80 (W1/2 ) 20.6 Hz) was assigned to the
H-3 axial proton geminally bearing an equatorial-oriented
hydroxy group. The hydroxymethine proton at δ 3.94
showed an HMBC correlation with the C-22 acetal carbon
at δ 112.0 and proton spin-coupling correlations with the
methylene protons at δ 2.10 and 1.84 (H2-24) and was
assigned to H-23. Consequently, the two quaternary car-
bons at δ 90.9 and 87.9 were concluded to each bear a
hydroxy group. The H2-15 and H-16 protons and the H-20
and Me-21 protons were observed as an ABX-spin system
at δ 2.60 (dd, J ) 12.8, 7.5 Hz) and 1.93 (dd, J ) 12.8, 6.4
Hz) and 4.94 (dd, J ) 7.5, 6.4 Hz) and an AM3-spin system
at δ 3.52 (q, J ) 7.1 Hz) and 1.35 (d, J ) 7.1 Hz). In the
HMBC spectrum, long-range correlations were observed
from H2-15 and H-16 to δ 87.9, from H-16, H-20, and Me-
21 to δ 90.9, and from the three-proton singlet at δ 1.35
due to Me-18 to both δ 87.9 and 90.9, allowing the δ 87.9
and 90.9 resonances to be assigned to C-14 and C-17,
respectively. The above data led to placement of the
hydroxy groups at C-3â, C-14, C-17, and C-23. NOE
correlations from H-16 to H-15R and H-26axial, Me-18 to
H-8, H-15â, and H-20, Me-19 to H-8 and H-11axial, and
H-23 to H-20, Me-21, and H-25, along with the proton spin-
coupling constants of H-23 (dd, J ) 11.2, 4.7 Hz) and
H-26axial (dd, J ) 10.6, 10.6 Hz), confirmed the B/C trans,
C/D trans, and D/E cis ring junctions and the 14R, 17R,
20R, 22R, 23S, and 25R configurations. The diglycoside
attached at C-3 of the aglycon of 3 was identified as O-R-
L-rhamnosyl-(1f2)-â-D-glucose, as in 1 and 2, on the basis
results of acid hydrolysis implied that the glycoside moiety
of 1 was composed of a terminal R-L-rhamnopyranosyl unit
(Rha) and a 2-substituted â-D-glucopyranosyl unit (Glc). In
the HMBC spectrum, the anomeric proton of Rha at δ 6.33
showed a long-range correlation with C-2 of Glc at δ 77.8,
for which the anomeric proton at δ 5.00 exhibited a
correlation with C-3 of the aglycon at δ 78.0. All of these
data were consistent with the structure (23S,24R,25S)-23,-
24-dihydroxyspirost-5-en-3â-yl O-R-L-rhamnopyranosyl-
(1f2)-â-D-glucopyranoside, which was given to 1.
Compound 2 was obtained as an amorphous solid with
a molecular formula of C39H62O15, as determined by the
data of the positive-ion HRESIMS (m/z 771.4191 [M + H]+).
1
The H NMR spectrum of 2 showed signals for two three-
proton doublets at δ 1.36 (J ) 7.2 Hz) and 0.69 (J ) 7.3
Hz) and two three-proton singlets at δ 1.13 and 0.99, as
well as two anomeric protons at δ 6.33 (d, J ) 1.0 Hz) and
4.97 (d, J ) 7.7 Hz), and the 13C NMR spectrum exhibited
an acetalic carbon signal at δ 112.0, suggesting that 2 is
also a spirostanol diglycoside. Enzymatic hydrolysis of 2
with naringinase resulted in the production of a new
steroidal sapogenin (2a: C27H42O6) and D-glucose and
L-rhamnose. The 1H NMR spectrum of 2a measured in
DMSO-d6 showed signals for four exchangeable protons at
δ 5.98, 5.74, 5.29, and 4.52, which disappeared on the
addition of HCl vapor, indicative of 2a having four hydroxy
groups. The gross structure of 2a was established in the
following spectroscopic data observation. The multiplet
proton signal centered at δ 3.78 (W1/2 ) 21.3 Hz) was shown
to be coupled with two methylene groups at δ 2.00 and 1.76
(H2-2) and δ 2.60 and 2.59 (H2-4) and was assigned to the
H-3 axial proton geminally bearing an equatorial-oriented
hydroxy group. The methyl singlet at δ 1.17 due to Me-18
3
exhibited JC,H correlations with the methine carbon at δ
74.1 and quaternary carbon at δ 93.4, each attached to a
hydroxy group. The hydroxymethine carbon at δ 74.1 was
associated with the one-bond coupled proton at δ 4.41,
which showed proton spin-coupling correlations with the
methylene protons at δ 1.81 and 1.74 (H2-11) and was
assigned to H-12. The H-20 and Me-21 protons were
observed as an AM3 spin system at δ 3.41 (q, J ) 7.2 Hz)
and 1.37 (d, J ) 7.2 Hz) and showed a long-range correla-
tions with the quaternary carbon at δ 93.4. Furthermore,
the oxymethine proton at δ 4.63 due to H-16 also showed
an HMBC correlation with δ 93.4. These data gave evidence
for the presence of a hydroxy group at C-12 and C-17. The
locus of the one remaining hydroxy group was determined
to be at C-23 by a long-range correlation between the
hydroxymethine proton at δ 3.92 and the C-22 acetal
carbon at δ 112.1 and by proton spin-coupling correlations
from δ 3.92 to the methylene protons at δ 2.09 and 1.84
(H2-24). NOE correlations from H-9 to H-14, H-12 to H2-
11, Me-18, and Me-21, H-16 to H-14, H-15R, and H-26axial,
1
of the H NMR, 13C NMR, and HMBC spectra of 3. Thus,
the structure of 3 was formulated as (23S,25R)-14R,17R,-
23-trihydroxyspirost-5-en-3â-yl O-R-L-rhamnopyranosyl-
(1f2)-â-D-glucopyranoside.
Although several spirostanols have been reported to
show potent cytotoxic activities for cultured tumor cell
lines,5-8 1-3, 2a, and 3a exhibited no apparent cytotoxic
activities against HSC-2 human squamous cell carcinoma

1118 Journal of Natural Products, 2005, Vol. 68, No. 7
Notes
Table 1. 1H and 13C NMR Data of 1, 2a, and 3a in Pyridine-d5
1
2a
J (Hz)
3a
J (Hz)
1H
J (Hz)
13C
1H
13C
1H
13C
1
2
eq
ax
eq
ax
1.71
0.95
2.11
1.85
3.92
2.78
2.70
37.4
1.77
1.13
2.00
1.76
3.78
2.60
2.59
37.7
1.83
1.13
2.05
1.78
3.80
2.60
38.0
ddd
13.3, 13.3, 3.4
ddd
13.5, 13.5, 3.5
30.1
32.4
32.5
3
4
br m
dd
dd
W1/2 ) 15.8
11.3, 3.0
11.3, 11.3
78.0
38.9
br m
dd
dd
W1/2 ) 21.3
12.3, 5.0
12.3, 12.3
71.2
43.4
br m
W1/2 ) 20.6
71.2
43.4
eq
ax
5
6
7
140.7
121.7
32.2
142.0
121.1
32.4
141.3
121.5
26.1
5.28
1.82
1.47
1.49
0.88
br d
4.9
5.36
2.00
1.71
1.73
1.84
br d
4.8
5.43
1.88
2.57
2.06
1.84
br d
4.3
eq
ax
8
9
10
11
31.5
50.2
37.1
21.0
32.8
43.7
36.7
29.6
36.1
43.5
37.2
20.1
eq
ax
eq
ax
1.40
1.35
1.72
1.12
1.81
1.74
4.41
1.65
1.60
1.41
2.59
12
40.1
br s
74.1
26.9
13
14
15
40.9
56.6
32.1
47.3
46.7
31.1
49.0
87.9
40.1
1.09
2.04
1.54
4.64
1.86
1.03
0.98
3.05
1.18
2.89
2.36
1.63
4.63
R
2.60
1.93
4.94
dd
dd
dd
12.8, 7.5
12.8, 6.4
7.5, 6.4
â
ddd
dd
12.9, 12.7, 6.3
7.6, 6.3
16
17
18
19
20
21
22
23
24
q-like
8.5
81.9
61.8
16.5
19.3
36.4
14.5
113.2
73.6
76.0
90.4
93.4
18.1
19.3
38.9
9.7
112.1
68.0
38.2
91.3
90.9
20.9
19.4
39.0
9.4
112.0
67.9
38.3
dd
s
s
8.5, 7.1
1.17
0.98
3.41
1.37
s
s
q
d
1.35
1.04
3.52
1.35
s
s
q
d
7.2
7.2
7.1
7.1
d
7.0
3.88
3.97
d
dd
9.4
9.4, 9.4
3.92
2.09
1.84
1.82
3.44
3.47
0.69
dd
11.2, 4.6
3.94
2.10
1.84
1.83
3.48
3.46
0.68
dd
11.2, 4.7
eq
ax
25
26
2.04
3.64
3.66
1.09
5.00
4.23
4.30
4.17
3.88
4.48
4.34
6.33
4.80
4.61
4.34
4.96
1.75
39.1
64.4
31.5
65.7
31.6
65.9
eq
ax
dd
d
d
11.3, 11.3
6.5
7.7
dd
d
11.2, 11.2
5.8
dd
d
10.6, 10.6
5.8
27
1′
2′
3′
4′
5′
6′
13.6
100.3
77.8
79.5
71.7
78.1
62.5
16.8
16.8
dd
dd
dd
ddd
dd
dd
d
dd
dd
dd
dq
d
9.1, 7.7
9.1, 9.1
9.3, 9.1
9.3, 5.0, 2.3
12.0, 2.3
12.0, 5.0
0.6
3.3, 0.6
9.3, 3.3
9.3, 9.3
9.3, 6.2
6.2
a
b
1′′
2′′
3′′
4′′
5′′
6′′
102.0
72.4
72.8
74.1
69.4
18.6
CCP PX-8010 controller (Tosoh), an RI-8010 detector (Tosoh)
or a Shodex OR-2 detector (Showa-Denko, Tokyo, Japan), and
a Rheodyne injection port. A Capcell Pak C18 UG120 column
(10 mm i.d. × 250 mm, 5 µm, Shiseido, Tokyo, Japan) was
employed for preparative HPLC.
Plant Material. Dioscorea polygonoides was collected at
Aranzazu, Caldas, Colombia, in October 1998. The plant was
identified by one of the authors (J.N.O.), and a voucher
specimen has been deposited in the herbarium of the Univer-
sidad de Antioquia, Medell´ın, Colombia (voucher no. HUA
132745).
Extraction and Isolation. A dried powder (1.0 kg) of D.
polygonoides tubers was extracted with MeOH (5 L) at 95 °C
for 3 h twice. After removal of the solvent by evaporation, the
viscous extract was partitioned between n-BuOH saturated
with H2O and H2O. The n-BuOH-soluble portion (9.2 g) was
subjected to vacuum-liquid chromatography on silica gel and
elution with CHCl3-MeOH (19:1), with increasing amounts
of MeOH (5% to 100%), to give eight fractions (fractions
I-VIII). Fraction V (1.5 g) was further separated by passage
over a silica gel column, using CHCl3-MeOH-H2O (14:5:1)
for elution, into five subfractions (fractions Va-Ve). Subfrac-
cells and HL-60 human promyelocytic leukemia cells (IC50
> 200 µM), respectively.
Experimental Section
General Experimental Procedures. Optical rotations
were measured using a JASCO DIP-360 (Tokyo, Japan)
automatic digital polarimeter. IR spectra were recorded on a
JASCO FT-IR 620 spectrophotometer. NMR spectra were
recorded on a Bruker DRX-500 spectrometer (500 MHz for 1H
NMR, Karlsruhe, Germany) using standard Bruker pulse
programs. Chemical shifts are given as δ values with reference
to tetramethylsilane (TMS) as internal standard. ESIMS data
were obtained on a Micromass LCT mass spectrometer
(Manchester, UK). Silica gel (Fuji-Silysia Chemical, Aichi,
Japan, or Merck, Darmstadt, Germany) and ODS silica gel
(Nacalai Tesque, Kyoto, Japan) were used for column chro-
matography. TLC was carried out on precoated Kieselgel 60
F254 (0.25 mm, Merck) and RP-18 F254 S (0.25 mm thick, Merck)
plates, and spots were visualized by spraying with 10% H2-
SO4 followed by heating. HPLC was performed by using a
system comprised of a CCPM pump (Tosoh, Tokyo, Japan), a

Notes
Journal of Natural Products, 2005, Vol. 68, No. 7 1119
tion Vc (0.22 g) was subjected to ODS silica gel column
chromatography, eluted with MeOH-H2O (16:7) and MeCN-
H2O (2:5), and then to preparative HPLC using MeCN-H2O
(2:5), to give 1 (12.6 mg), 2 (74.4 mg), and 3 (34.1 mg).
26
Compound 1: amorphous solid; [R]D -114.0° (c 0.10,
MeOH); IR (film) νmax 3379 (OH), 2917 and 2849 (CH), 1044
1
cm-1; H and 13C NMR, see Table 1; HRESIMS m/z 755.4213
[M + H]+ (calcd for C39H63O14, 755.4218).
Acid Hydrolysis of 1. A solution of 1 (4.8 mg) in 1.0 M
HCl (dioxane-H2O, 1:1, 3 mL) was heated at 95 °C for 1 h
under an Ar atmosphere. On cooling, the reaction mixture was
neutralized by passage through an Amberlite IRA-93 ZU
(Organo, Tokyo, Japan) column and then passed through a
Sep-Pak C18 cartridge (Waters, Milford, MA), eluted with 10%
MeOH followed by MeOH. The 10% MeOH eluate fraction (1.2
mg) was analyzed by HPLC under the following conditions:
column, Capcell Pak NH2 UG80 (4.6 mm i.d. × 250 mm, 5 µm,
Shiseido, Tokyo, Japan); solvent, MeCN-H2O (17:3); flow rate,
1.0 mL/min; detection, RI and OR. Identification of L-rhamnose
and D-glucose was carried out by comparison of their retention
times and optical rotations with those of authentic samples:
tR (min), 6.39 (L-rhamnose, negative optical rotation), 11.78
(D-glucose, positive optical rotation).
Acetylation of 1. Compound 1 (1.9 mg) was treated with
Ac2O (1 mL) in pyridine (1 mL) at room temperature for 20 h.
After addition of H2O, the reaction mixture was extracted with
Et2O and the Et2O phase was chromatographed on silica gel,
eluted with hexane-Me2CO (3:1), to afford 1a (1.6 mg).
Figure 1. Partial HMBC (arrows) and NOE (curved lines) correlations
of 1, 2a, and 3a.
24
Compound 2a: amorphous solid; [R]D -102.0° (c 0.10,
MeOH); IR (film) νmax 3352 (OH), 2953, 2927 and 2861 (CH),
25
1091, 1057 cm-1; 1H NMR (DMSO-d6) δ 5.98 (s), 5.74 (s), 5.29
Compound 1a: amorphous solid; [R]D -56.0° (c 0.10,
(d, J ) 5.0 Hz), 4.52 (d, J ) 7.7 Hz); H (pyridine-d5) and 13C
MeOH); IR νmax (film) 2956, 2918 and 2849 (CH), 1748 (CdO)
1
1
cm-1; H NMR (pyridine-d5) δ 5.83 (1H, dd, J ) 9.5, 9.5 Hz,
NMR, see Table 1; HRESIMS m/z 463.3069 [M + H]+ (calcd
H-3′), 5.80 (1H, dd, J ) 10.1, 3.4 Hz, H-3′′), 5.68 (1H, dd, J )
10.1, 10.1 Hz, H-4′′), 5.61 (1H, dd, J ) 3.4, 1.5 Hz, H-2′′), 5.51
(1H, dd, J ) 10.3, 9.8 Hz, H-24), 5.45 (1H, d, J ) 1.5 Hz, H-1′′),
5.44 (1H, dd, J ) 9.8, 9.5 Hz, H-4′), 5.43 (1H, br d, J ) 5.0 Hz,
H-6), 5.36 (1H, d, J ) 9.8 Hz, H-23), 5.07 (1H, d, J ) 7.8 Hz,
H-1′), 4.93 (1H, dq, J ) 10.1, 6.3 Hz, H-5′′), 4.65 (1H, dd, J )
12.3, 4.5 Hz, H-6′a), 4.56 (1H, q-like, J ) 8.5 Hz, H-16), 4.43
(1H, dd, J ) 12.3, 2.2 Hz, H-6′b), 4.15 (1H, ddd, J ) 9.8, 4.5,
2.2 Hz, H-5′), 4.12 (dd, J ) 9.5, 7.8 Hz, H-2′), 3.94 (1H, br m,
W1/2 ) 15.8 Hz, H-3), 3.60 (1H, dd, J ) 11.3, 5.9 Hz, H-26eq),
3.57 (1H, dd, J ) 11.3, 11.3 Hz, H-26ax), 2.21, 2.17, 2.15, 2.08,
2.05 × 2, 2.03, 2.02 (each 3H, s, Ac × 8), 1.51 (3H, d, J ) 6.3
Hz, Me-6′′), 1.20 (3H, d, J ) 7.0 Hz, Me-21), 1.11 (3H, s, Me-
18), 0.89 (3H, s, Me-19), 0.80 (3H, d, J ) 6.6 Hz, Me-27).
for C27H43O6, 463.3060).
26
Compound 3: amorphous solid; [R]D -84.0° (c 0.10,
MeOH); IR (film) νmax 3393 (OH), 2957, 2932 and 2874 (CH),
1
1055 cm-1; H NMR (pyridine-d5) δ 6.33 (1H, d, J ) 1.0 Hz,
H-1′′), 5.33 (1H, br d, J ) 4.8 Hz, H-6), 4.98 (1H, d, J ) 7.7
Hz, H-1′), 4.94 (1H, m, H-16), 3.94 (1H, dd, J ) 11.2, 4.7 Hz,
H-23), 3.87 (1H, br m, W1/2 ) 18.0 Hz, H-3), 3.47 (2H, m, H2-
26), 1.75 (3H, d, J ) 6.2 Hz, Me-6′′), 1.34 (3H, d, J ) 7.3 Hz,
Me-21), 1.31 (3H, s, Me-18), 1.04 (3H, s, Me-19), 0.68 (3H, d,
J ) 5.9 Hz, Me-27); 13C NMR (pyridine-d5) δ 37.7 (C-1), 30.1
(C-2), 77.8 (C-3), 38.9 (C-4), 140.1 (C-5), 122.2 (C-6), 26.1 (C-
7), 36.0 (C-8), 43.4 (C-9), 37.3 (C-10), 20.0 (C-11), 26.8 (C-12),
48.9 (C-13), 87.9 (C-14), 40.1 (C-15), 91.2 (C-16), 90.8 (C-17),
20.8 (C-18), 19.3 (C-19), 38.9 (C-20), 9.4 (C-21), 112.0 (C-22),
67.9 (C-23), 38.3 (C-24), 31.5 (C-25), 65.9 (C-26), 16.8 (C-27),
100.2 (C-1′), 77.8 (C-2′), 79.5 (C-3′), 71.7 (C-4′), 78.2 (C-5′), 62.5
(C-6′), 102.0 (C-1′′), 72.4 (C-2′′), 72.8 (C-3′′), 74.1 (C-4′′), 69.4
(C-5′′), 18.6 (C-6′′); HRESIMS m/z 793.3992 [M + Na]+ (calcd
for C39H62O15Na, 793.3986).
27
Compound 2: amorphous solid; [R]D -98.0° (c 0.10,
MeOH); IR (film) νmax 3392 (OH), 2954, 2932 and 2905 (CH),
1
1052 cm-1; H NMR (pyridine-d5) δ 6.33 (1H, d, J ) 1.0 Hz,
H-1′′), 5.28 (1H, br d, J ) 4.8 Hz, H-6), 4.97 (1H, d, J ) 7.7
Hz, H-1′), 4.63 (1H, dd, J ) 7.5, 6.3 Hz, H-16), 4.38 (1H, br s,
H-12), 3.89 (1H, br m, W1/2 ) 17.8 Hz, H-3), 3.91 (1H, m, H-23),
3.46 (2H, m, H2-26), 1.74 (3H, d, J ) 6.2 Hz, Me-6′′), 1.36 (3H,
d, J ) 7.2 Hz, Me-27), 1.13 (3H, s, Me-18), 0.99 (3H, s, Me-
19), 0.69 (3H, d, J ) 7.3 Hz, Me-21); 13C NMR (pyridine-d5) δ
37.3 (C-1), 30.1 (C-2), 77.8 (C-3), 38.9 (C-4), 140.8 (C-5), 121.8
(C-6), 32.4 (C-7), 32.7 (C-8), 43.6 (C-9), 36.8 (C-10), 29.5 (C-
11), 74.0 (C-12), 47.3 (C-13), 46.5 (C-14), 31.1 (C-15), 90.3 (C-
16), 93.3 (C-17), 18.0 (C-18), 19.1 (C-19), 38.9 (C-20), 9.7 (C-
21), 112.0 (C-22), 68.0 (C-23), 38.2 (C-24), 31.5 (C-25), 65.7 (C-
26), 16.8 (C-27), 100.2 (C-1′), 77.8 (C-2′), 79.5 (C-3′), 71.7 (C-
4′), 78.1 (C-5′), 62.5 (C-6′), 102.0 (C-1′′), 72.4 (C-2′′), 72.8 (C-
3′′), 74.1 (C-4′′), 69.4 (C-5′′), 18.6 (C-6′′); HRESIMS m/z
771.4191 [M + H]+ (calcd for C39H63O15, 771.4167).
Enzymatic Hydrolysis of 3. Compound 3 (15.2 mg) was
subjected to enzymatic hydrolysis using naringinase as de-
scribed for 2 to give an aglycon (3a) (9.1 mg) and a sugar
fraction. HPLC analysis of the sugar fraction under the same
conditions as for 1 showed the presence of L-rhamnose and
D-glucose: tR (min), 6.45 (L-rhamnose, negative optical rota-
tion), 11.89 (D-glucose, positive optical rotation).
24
Compound 3a: amorphous solid; [R]D -72.0° (c 0.10,
MeOH); IR (film) νmax 3357 (OH), 2927 and 2871 (CH), 1058
cm-1; 1H NMR (DMSO-d6) δ 5.28 (d, J ) 4.8 Hz), 4.94 (s), 4.59
1
(s), 4.51 (d, J ) 7.9 Hz); H (pyridine-d5) and 13C NMR, see
Table 1; HRESIMS m/z 485.2824 [M + Na]+ (calcd for
C27H42O6Na, 485.2879).
Enzymatic Hydrolysis of 2. Compound 2 (20.9 mg) was
treated with naringinase (EC 232-962-4; Sigma, St. Louis, MO)
(80 mg) in AcOH-AcOK buffer (pH 4.3, 10 mL) at room
temperature for 72 h. The reaction mixture was passed
through a Sep-Pak C18 cartridge (Waters, Milford, MA), eluted
with 20% MeOH followed by MeOH. The MeOH eluate fraction
was purified by silica gel column chromatography, eluted with
CHCl3-MeOH (20:1), to afford 2a (12.4 mg). HPLC analysis
of the 20% MeOH eluate fraction under the same conditions
as used for 1 showed the presence of L-rhamnose and D-
glucose: tR (min), 6.41 (L-rhamnose, negative optical rotation),
11.87 (D-glucose, positive optical rotation).
HSC-2 and HL-60 Cell Culture Assay. The cell growth
was measured with an MTT reduction assay procedure as
described in previous papers.7,8
Acknowledgment. We would like to thank COLCIEN-
CIAS and the Universidad Tecnolo´gica de Pereira for financial
support for project No. 1110-05-515-96; 202-96.
References and Notes
(1) Hostettmann, K.; Marston, A. Chemistry and Pharmacology of
Natural Products: Saponins; Cambridge University Press: Cam-
bridge, U.K., 1995.

1120 Journal of Natural Products, 2005, Vol. 68, No. 7
Notes
(2) Mahato, S. B.; Ganguly, A. N.; Sahu, N. P. Phytochemistry 1982, 21,
959-978.
(6) Candra, E.; Matsunaga, K.; Fujuwara, H.; Mimaki, Y.; Sashida, Y.;
Yamakuni, T.; Ohizumi, Y. Can. J. Physiol. Pharmacol. 2001, 79,
953-958.
(3) Mimaki, Y.; Sashida, Y.; Nakamura, O.; Nikaido, T.; Ohmoto, T.
Phytochemistry 1993, 33, 675-682.
(7) Mimaki, Y.; Yokosuka, A.; Kuroda, M.; Sashida, Y. Biol. Pharm. Bull.
2001, 24, 1286-1289.
(4) Mimaki, Y.; Nakamura, O.; Sashida, Y.; Nikaido, T.; Ohmoto, T.
Phytochemistry 1995, 38, 1279-1286.
(5) Mimaki, Y.; Kuroda, M.; Obata, Y.; Sashida, Y.; Kitahara, M.; Yasuda,
A.; Naoi, N.; Xu, Z. W.; Li, M. R.; Lao, A. N. Nat. Prod. Lett. 2000,
14, 357-364.
(8) Mimaki, Y.; Watanabe, K.; Ando, Y.; Sakuma, C.; Sashida, Y.; Furuya,
S.; Sakagami, H. J. Nat. Prod. 2001, 64, 17-22.
NP0580356