Pregnane glycosides from the bark of Marsdenia cundurango and their cytotoxic activity

Article abstract of DOI:10.1007/s11418-018-1248-0

Seven new pregnane glycosides (1–7) and eight known compounds (8–15) were isolated from the bark of Marsdenia cundurango (Asclepiadaceae). The structures of 1–7 were determined by spectroscopic analysis, including two-dimension NMR spectroscopy, chemical transformations, and chromatographic analysis of the hydrolyzed products. The isolated compounds 1–15 were evaluated for their cytotoxic activity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and TIG-3 normal human lung cells, including apoptosis-inducing activity of a representative pregnane glycoside in HL-60 cells.

Full text of DOI:10.1007/s11418-018-1248-0

Journal of Natural Medicines
https://doi.org/10.1007/s11418-018-1248-0
ORIGINAL PAPER
Pregnane glycosides from the bark of Marsdenia cundurango and their
cytotoxic activity
Satoru Tatsuno1 · Akihito Yokosuka1 · Fusako Hatsuma1 · Yuto Mashiko1 · Yoshihiro Mimaki1
Received: 30 July 2018 / Accepted: 30 August 2018
© The Japanese Society of Pharmacognosy and Springer Japan KK, part of Springer Nature 2018
Abstract
Seven new pregnane glycosides (1–7) and eight known compounds (8–15) were isolated from the bark of Marsdenia cun-
durango (Asclepiadaceae). The structures of 1–7 were determined by spectroscopic analysis, including two-dimension NMR
spectroscopy, chemical transformations, and chromatographic analysis of the hydrolyzed products. The isolated compounds
1–15 were evaluated for their cytotoxic activity against HL-60 human leukemia cells, A549 human lung adenocarcinoma
cells, and TIG-3 normal human lung cells, including apoptosis-inducing activity of a representative pregnane glycoside in
HL-60 cells.
Keywords Marsdenia cundurango · Asclepiadaceae · Pregnane glycosides · Cytotoxic activity · HL-60 cell line · A549 cell
line · TIG-3 cell line · Apoptosis
Introduction
glycosides (1–15), including seven new compounds (1–7).
The structures of 1–7 were determined by spectroscopic
analysis, including two-dimension (2D) NMR spectroscopic
data, chemical transformation, and chromatographic analysis
of the hydrolyzed products. The isolated compounds (1–15)
were evaluated for their cytotoxic activity against HL-60
human promyelocytic leukemia cells, A549 human lung
adenocarcinoma cells, and TIG-3 normal human diploid
lung cells, as well as for the apoptosis-inducing activity of a
representative pregnane glycoside in HL-60 cells.
The bark of Marsdenia cundurango Reich. (Asclepia-
daceae), referred to as Condurango Cortex in the Japanese
Pharmacopoeia (17th edition), has been used as a remedy
for stomach complaints [1]. Several pregnane derivatives and
glycosides, named condurangogenins and condurangoglyco-
sides, have been isolated from M. cundurango bark, and their
cytotoxicity, anticancer activity, and diferentiation-inducing
activity are well documented [2–11]. In our continued phy-
tochemical studies on higher plants, we have isolated many
bioactive steroidal glycosides with an extremely diverse
range of structures, including pregnane glycosides, carde-
nolide glycosides, and bufadienolide glycosides [12–14]. As
part of our ongoing study of bioactive steroidal compounds
from natural sources, we investigated the pregnane glyco-
sides of the bark of M. cundurango and isolated 15 pregnane
Results and discussion
The bark of M. cundurango (5.0 kg) was extracted in hot
MeOH. After removal of the solvent, the MeOH extract was
subjected to column chromatography (CC) using Diaion
HP-20 porous-polymer polystyrene resin, silica gel, and
octadecylsilanized (ODS) silica gel to yield compounds
1–15 (Fig. 1). The structures of the known pregnane glyco-
sides (8–15) [8, 9, 15] identifed are shown in Fig. 1.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11418-018-1248-0) contains
supplementary material, which is available to authorized users.
Compound 1 was obtained as an amorphous solid and
had a molecular formula of C42H72O15, as determined from
high-resolution electrospray ionization time-of-fight-MS
(HRESI-TOF-MS) (m/z: 839.4759 [M+Na]+) and 13C-
* Akihito Yokosuka
yokosuka@toyaku.ac.jp
1
Department of Medicinal Pharmacognosy, School
1
NMR data. The H- and 13C-NMR spectra of 1 suggested
of Pharmacy, Tokyo University of Pharmacy and Life
Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392,
Japan
that it was the corresponding deacyl pregnane glycoside of
Vol.:(0123456789)
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Journal of Natural Medicines
OH
OH
OR4
O
HO
OH
OH
O
R3O
R3O
H
H
R2O
R2O
Ac =
Bz=
OH
OH
H
H
H
O
H
OH
S2O
R1O
R1O
H
H
14
R1
S4
S5
S1
S6
S7
R2
R3
R1
S1
S2
S3
S1
S1
S1
S1
S2
R2
H
H
H
H
Cin
Ac
Ac
Ac
R3
H
H
H
H
R4
H
H
H
Glc
H
H
H
H
Ac Cin
Ac Cin
Ac Cin
Ac Cin
Ac Cin
6
7
11
12
13
1
2
3
4
O
O
H
Cin-O
Ac-O
Cin =
H
H
5
8
9
H
OH
Bz
Cin
Cin
HO
HO
S2O
H
O
Glc= HO
10
15
OH
O
O
O
O
O
O
S1= HO
O
HO
O
O
O
O
O
O
O
O
O
O
O
O
S5=
O
O
OH
OH
O
O
O
O
HO
O
O
HO
O
O
O
O
HO
HO
O
S2=
HO
HO
O
O
S6=
O
O
OH
OH
OH
O
O
O
HO
HO
O
O
O
O
S3= HO
O
O
O
O
O
O
S7=
O
O
O
O
O
O
OH
OH
OH
O
O
HO
O
O
O
O
O
HO
S4=
O
O
Fig. 1 Structures of the pregnane glycosides 1–15 isolated from the bark of Marsdenia cundurango Reich. (Asclepiadaceae)
δ
C 82.9
δ
C 83.6
9. Acid hydrolysis of 1 using 0.025 M HCl in dioxane–H2O
(1:1) gave d-cymarose (Cym), O-(6-deoxy-3-O-methyl-β-
d-allopyranosyl)-(1 → 4)-d-oleandropyranose [16] and a
pregnane derivative, which was identifed as 5α-pregnane-
3β,11α,12β,14β,20-pentol (1a) [4, 17]. Comparison of the
13C-NMR spectrum of 1a with those of (20S)- and (20R)-
isolineolin-3,12-diacetate-20-ol allowed the configura-
tion at C-20 of 1a to be assigned as R [7]. The results of
H
δ
O
C 102.0
δ
C 101.9
H
δ
C 95.9
C4''
O
C4'
O
HO
O
C3
O
O
C1'''
H
C1''
H
C1'
H
H
H
HO
O
O
δ
C 76.6
δH 5.31
δ
H 4.68
δ
H 5.31
Fig. 2 Key HMBC correlations of isolated compound 1
acid hydrolysis and analysis of the H-NMR and H–1H
COSY spectra of 1 indicated that the sugar moiety of 1
was composed of a C-4-substituted β-d-cymaropyranosyl
(4C1) unit (Cym), a C-4 substituted β-d-oleandropyranosyl
(4C1) unit (Ole), and a terminal 6-deoxy-3-O-methyl-β-
d-allopyranosyl (4C1) unit (All); the O-(6-deoxy-3-O-
methyl-β-d-allopyranosyl)-(1 → 4)-O-d-oleandropyrano-
syl-(1→4)-β-d-cymaropyranosyl sequence and its linkage
to C-3 of the aglycone were confrmed by the long-range
correlations between H-1 of All at δH 5.31 and C-4 of
Ole at δC 82.9, between H-1 of Ole at δH 4.68 and C-4
of Cym at δC 83.6, and between H-1 of Cym at δH 5.31
and C-3 of the aglycone at δC 76.6 in the HMBC spec-
trum of 1 (Fig. 2). Thus, the structure of 1 was elucidated
as (20R)-11α,12β,14β,20-tetrahydroxy-5α-pregnan-3β-yl
1
1
O-(6-deoxy-3-O-methyl-β-d-allopyranosyl)-(1 → 4)-O-β-
d-oleandropyranoyl-(1→4)-β-d-cymaropyranoside.
HRESI-TOF-MS and 13C-NMR data showed that
the molecular formulae of 2 and 3 were C48H82O20 (m/z
1001.5293 [M+Na]+) and C41H70O15 (m/z 761.4116
[M+Na]+), respectively. The 1H- and 13C-NMR spectra of
2 and 3 indicated that each aglycone moiety was the same as
that of 1; however, the sugar residue attached to C-3 of the
aglycone was slightly diferent from that of 1. The molecular
formula of 2 was larger than that of 1 by C6H10O5. Enzy-
matic hydrolysis of 2 using β-d-glucosidase produced 1 and
d-glucose (Glc). In the HMBC spectrum of 2, long-range
correlations were observed between H-1 of Glc at δH 4.97
and C-4 of All at δC 83.3, between H-1 of All at δH 5.24
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Journal of Natural Medicines
and C-4 of Ole at δC 83.0, between H-1 of Ole at δH 4.64
and C-4 of Cym at δC 83.4, and between H-1 of Cym at δH
5.27 and C-3 of the aglycone at δC 76.6. Thus, the structure
of 2 was assigned as (20R)-11α,12β,14β,20-tetrahydroxy-
5α-pregnan-3β-yl O-β-d-glucopyranosyl-(1 → 4)-O-(6-
deoxy-3-O-methyl-β-d-allopyranosyl)-(1 → 4)-O-β-d-
oleandropyranosyl-(1→4)-β-d-cymaropyranoside.
863.4201 [M+Na]+) and C59H86O21 (m/z 1153.5536
[M+Na]+), respectively. The 1H- and 13C-NMR spectra of 6
and 7 possessed the same diacylated aglycone as the known
compounds 11–13, 11α-acetoxy-12β-[((E)-cinnamoyl)oxy]-
3β,8β,14β-trihydroxypregn-5-en-20-one (condurangogenin
E), but the sugar moieties of 6 and 7 were not coincident
with those of the compounds isolated in the current study.
The 1H-NMR spectrum of 6 contained signals for two ano-
meric protons at δH 5.29 (br d, J =9.5 Hz) and 5.08 (br d,
J=9.6 Hz). Acid hydrolysis of 6 yielded only d-cymarose
(Cym). These data suggested that 6 was condurangogenin E
dicymaroside (Cym I and Cym II). In the HMBC spectrum
of 6, correlation peaks were observed between H-1 of Cym
II at δH 5.08 and C-4 of Cym I at δC 83.2, and between H-1
of Cym I at δH 5.29 and C-3 of the aglycone at δC 77.7. Thus,
the structure of 6 was elucidated as 11α-acetoxy-12β-[((E)-
cinnamoyl)oxy]-8β,14β-dihydroxy-20-oxopregn-5-en-3β-yl
O-β-d-cymaropyranosyl-(1→4)-β-d-cymaropyranoside.
Acid hydrolysis of 3 yielded 1a, d-digitoxose (Dig), and
O-(6-deoxy-3-O-methyl-β-d-allopyranosyl)-(1→4)-d-ole-
andropyranose. The HMBC spectrum showed correlation
peaks between H-1 of All at δH 5.30 and C-4 of Ole at δC
82.9, between H-1 of Ole at δH 4.73 and C-4 of Dig at δC
83.7, and between H-1 of Dig at δH 5.51 and C-3 of the agly-
cone at δC 76.7. The structure of 3 was therefore determined
to be (20R)-11α,12β,14β,20-tetrahydroxy-5α-pregnan-3β-yl
O-(6-deoxy-3-O-methyl-β-d-allopyranosyl)-(1→4)-O-β-d-
oleandropyranosyl-(1→4)-β-d-digitoxopyranoside.
HRESI-TOF-MS and 13C-NMR data showed that
the molecular formulae of 4 and 5 were C48H82O20 (m/z
1001.5300 [M+Na]+ and C51H78O16 (m/z 969.5184
[M+Na]+), respectively. Analysis of the 1H- and 13C-NMR
spectra suggested that 4 and 5 were essentially analogous
to 1, but difered structurally from 1 with a substituent on
1
The H-NMR spectrum of 7 exhibited signals for four
anomeric protons at δH 5.32 (br d, J = 9.4 Hz), 5.30 (d,
J=8.1 Hz), 5.29 (dd, J=9.5, 1.7 Hz), and 4.72 (dd, J=9.6,
1.3 Hz). Acid hydrolysis of 7 produced d-cymarose (Cym),
d-digitoxose (Dig), and O-(6-deoxy-3-O-methyl-β-d-
allopyranosyl)-(1→4)-d-oleandropyranose. In the HMBC
spectrum, correlation peaks were detectable between H-1 of
All at δH 5.30 and C-4 of Ole at δC 83.0, between H-1 of Ole
at δH 4.72 and C-4 of Dig at δC 83.2, between H-1 of Dig at
δH 5.32 and C-4 of Cym at δC 83.3, and between H-1 of Cym
at δH 5.29 and C-3 of the aglycone at δC 77.7. The struc-
ture of 7 was thus determined to be 11α-acetoxy-12β-[((E)-
cinnamoyl)oxy]-8β,14β-dihydroxy-20-oxopregn-5-en-3β-yl
O-(6-deoxy-3-O-methyl-β-d-allopyranosyl)-(1→4)-O-β-d-
oleandropyranosyl-(1 → 4)-O-β-d-digitoxopyranosyl-β-d-
cymaropyranoside.
1
the aglycone moiety. The H- and 13C-NMR spectra of 4
were indicative of the presence of a β-d-glucopyranosyl
group (Glc) [δH 4.98 (1H, d, J = 8.6 Hz); δC 101.6, 75.2,
78.8, 72.0, 78.4, and 63.2 (Glc-1–6)]. The 13C-NMR sig-
nal at C-20 of 4 was shifted downfeld than that of 1 by
7.0 ppm. In the HMBC spectrum of 4, a long-range cor-
relation was observed between H-1 of Glc at δH 4.98 and
C-20 of the aglycone at δC 77.3. Thus, 4 was revealed to
be a bisdesmosidic pregnane glycoside, and its structure
was determined to be (20R)-[20-(β-d-glucopyranosyl)
oxy]-11α,12β,14β-trihydroxy-5α-pregnan-3β-yl O-(6-
deoxy-3-O-methyl-β-d-allopyranosyl)-(1 → 4)-O-β-d-
oleandropyranosyl-(1→4)-β-d-cymaropyranoside.
The isolated compounds were evaluated for their cyto-
toxic activity against HL-60 human leukemia cells and A549
human lung adenocarcinoma cells (Table 1). Compounds
5, 6, 7, 11, and 12 exhibited relatively potent and tumor-
selective cytotoxicity against HL-60 cells, with IC50 values
of between 3.75 and 6.09 µM. A549 solid cells were less
sensitive to these compounds, with IC50 values of between
12.2 and 16.9 µM.
For compound 5, the signals arising from an (E)-cin-
1
namoyl group were detected in the H- and 13C-NMR
spectra [δH 7.92 (1H, d, J = 16.0 Hz, Cin), 7.50 (2H, m,
Cin), 7.33 (1H, m, Cin), 7.32 (2H, m, Cin), and 6.77 (1H,
d, J=16.0 Hz, Cin); δc 167.0, 120.2, 144.4, 135.0, 128.4,
129.1, and 130.4]. The 13C-NMR signals assigned to
C-11 and C-12 of 5 were not coincident with those of 1.
The ester linkage at the aglycone C-11 hydroxy group was
formed from (E)-cinnamic acid, as was evident from the
long-range correlation between the H-11 proton at δH 5.81
and the carbonyl carbon of the (E)-cinnamoyl group at δC
167.0. The structure of 5 was assigned as (20R)-11α-[((E)-
cinnamoyl)oxy]-12β,14β,20-trihydroxy-5α-pregnan-3β-yl
O-(6-deoxy-3-O-methyl-β-d-allopyranosyl)-(1 → 4)-O-β-
d-oleandropyranosyl-(1→4)-β-d-cymaropyranoside.
HRESI-TOF-MS and 13C-NMR data showed that
the molecular formulae of 6 and 7 were C46H64O14 (m/z
The representative new pregnane glycoside (6) isolated
from M. cundurango bark was evaluated for apoptosis-induc-
ing activity in HL-60 cells. In cells treated with 6 (20 µM),
nuclear chromatin condensation and apoptotic bodies were
observed in 4′,6-diamidino-2-phenylindole dihydrochloride
(DAPI)-stained samples under a fuorescence microscope
(Fig. 3). The disruption of mitochondrial membrane poten-
tial (∆Ψm) occurs in the early stages of apoptosis. When
HL-60 cells were treated with 6 (20 µM) for 6 h, the loss of
∆Ψm was detected by using MitoCapture™ reagents (Fig. 4).
Caspase-3, which plays an important role in the apoptotic
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Journal of Natural Medicines
Table 1 Cytotoxic activity of compounds 1–15 and cisplatin against
HL-60 human leukemia cells, A549 human lung adenocarcinoma
cells, and TIG-3 normal human lung cells
*
Compounds
IC50 (μM)a
*
HL-60
A549
TIG-3
1
22.0 1.61
>25
–
>25
>25
_
_
12.2 1.34
15.7 0.47
16.3 0.63
>25
>25
>25
16.9 0.75
12.8 1.87
>25
>25
–
>25
>25
_
2
a
3
4
_
_
C
6
E
5
4.18 0.24
4.64 0.53
5.56 0.12
14.1 2.16
8.38 0.91
>25
6.09 0.54
3.75 0.31
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
–
6
7
Fig. 5 Caspase-3 activity in the lysates of cells treated with 6 or
etoposide. HL-60 cells were incubated with 20 μM of 6 (6) or 15 μM
of etoposide (E) for 16 h, and caspase-3 activity in the lysate was
measured using a caspase-3 colorimetric assay kit. The data are pre-
sented as the mean standard error of the mean of three experiments.
Asterisk indicates that caspase-3 activity in the treated cells was sig-
nifcantly diferent at p<0.001 from that of the control (C) cells
8
9
10
11
12
13
14
>25
–
1.96 0.16
signaling pathways and is the enzyme that executes apop-
tosis, was activated when HL-60 cells were treated with 6
(20 µM) for 16 h (Fig. 5). These data suggest that 6 induced
apoptotic cell death in HL-60 cells through a loss of mito-
chondrial membrane potential, followed by the activation
of caspase-3.
15
Cisplatin
1.85 0.38
4.79 1.79
aThe data are presented as the mean standard error of the mean
(SEM) of three experiments performed in triplicate
aNot determined owing to low yields
6
Conclusion
Control
Etoposide
Seven new pregnane glycosides (1–7) and eight known
compounds (8–15) were isolated from the bark of M. cun-
durango. The structures of 1–7 were determined by spec-
troscopic analyses, including 2D NMR spectroscopic data,
chemical transformations, and chromatographic analysis of
the hydrolyzed products. The isolated compounds were eval-
uated for cytotoxic activity against HL-60 and A549 cells
(Table 1). Several pregnane glycosides showed relatively
potent and tumor-selective cytotoxicity against HL-60 cells.
The representative new pregnane glycoside 6 was revealed
to induce apoptotic cell death in HL-60 cells, which was
identifed by the typical indicators of apoptosis, such as frag-
mented and condensed nuclear chromatins and the loss of
mitochondrial membrane potential followed by the activa-
tion of caspase-3.
Fig. 3 Morphology of HL-60 human leukemia cells treated with
isolated compound 6 or etoposide. HL-60 cells were stained with
4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) after treat-
ment with 20 μM of 6 or 15 μM of etoposide for 24 h, and observed
under a fuorescence microscope
6
Control
Cisplatin
Experimental
General experimental procedures
Fig. 4 Mitochondrial membrane appearance of HL-60 cells treated
with 6 or cisplatin. HL-60 cells were stained with MitoCaptureTM
after treatment with 20 μM of 6 or 30 μM of cisplatin for 6 h, and
observed under a fuorecence microscope
Optical rotations were measured using a model P-1030
automatic digital polarimeter (JASCO, Tokyo, Japan). UV
and IR spectra were recorded on a JASCO V-630 UV–Vis
1 3

Journal of Natural Medicines
spectrophotometer and JASCO FT-IR 620 spectropho-
tometer, respectively. NMR spectral data were obtained
(5 L), EtOH (5 L), and EtOAc (2.5 L). The MeOH elu-
ate (130 g) was subjected to CC using silica gel (60 mm
i.d.×300 mm), eluted with CHCl3–MeOH–H2O (19:1:0.1,
9:1:0.1, 6:1:0.1, 4:1:0.1, 2:1:0.1) and MeOH alone, to
yield 12 fractions (A–L). Fraction B was chromatographed
on silica gel eluted with CHCl3–MeOH–H2O (9:1:0.1,
6:1:0.1, 4:1:0.1, 2:1:0.1, 1:1) and ODS silica gel eluted
with MeCN–H2O (3:2) to produce compounds 3 (12.1 mg)
and 15 (9.7 mg). Fraction C was separated by silica gel CC
eluted with CHCl3–MeOH (50:1, 10:1) and by ODS silica
gel CC eluted with MeCN–H2O (1:1) to produce compounds
5 (8.7 mg), 6 (9.0 mg), 7 (9.8 mg), 8 (10.3 mg), 9 (47.2 mg),
11 (24.3 mg), and 12 (16.4 mg). Fraction E was separated
by ODS silica gel CC eluted with MeCN–H2O (1:2; 1:1)
to produce compounds 1 (25.3 mg), 10 (10.4 mg), and 13
(16.4 mg). Fraction F was separated by ODS silica gel CC
eluted with MeCN–H2O (1:4; 1:3, 1:2) to obtain compounds
4 (2.1 mg), 10 (23.4 mg), and 14 (2.8 mg). Fraction I was
separated by ODS silica gel CC eluted with MeCN–H2O
(1:4; 1:3, 1:2) to give compound 2 (10.1 mg).
1
on a DRX-500 spectrometer (500 MHz for H-NMR;
Bruker, Karlsruhe, Germany) using standard Bruker pulse
programs at 300 K. Chemical shifts were presented as δ
values with reference to tetramethylsilane as an internal
standard. HRESI-TOF-MS data were recorded on an LCT
mass spectrometer (Waters-Micromass, Manchester, UK).
Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan), silica
gel (Fuji-Silysia Chemical, Aichi, Japan), ODS silica gel
(Nacalai Tesque, Kyoto, Japan), and Amberlite IRA-96SB
(Organo, Tokyo, Japan) were used for CC. TLC was per-
formed on precoated silica gel 60 F254 (thickness 0.25 mm;
Merck, Darmstadt, Germany) and RP18 F254S plates (thick-
ness 0.25 mm; Merck), and spots were visualized by spray-
ing the plates with 10% H2SO4 aqueous solution followed
by heating. HPLC was performed with a system consisting
of a CCPM pump (Tosoh, Tokyo, Japan), a Shodex OR-2
detector (Showa-Denko, Tokyo, Japan), and a Rheodyne™
injection port (Thermo Fisher Scientifc, Waltham, MA,
USA). HL-60 cells (JCRB 0085), A549 cells (JCRB 0076),
and TIG-3 cells (JCRB 0510) were obtained from the Japa-
nese Collection of Research Bioresources Cell Bank (JCRB;
Osaka, Japan). The following materials and reagents were
used for the cell culture assays: a Spectra Classic microplate
reader (Tecan, Salzburg, Austria), a CKX41 fuorescence
microscope (Olympus, Tokyo, Japan), 96-well fat-bottom
plates (Iwaki Glass, Chiba, Japan), fetal bovine serum (FBS)
(Nichirei Biosciences, Tokyo, Japan), 0.25% trypsin–EDTA
solution, RPMI-1640 medium, minimum essential medium
(MEM), cisplatin, etoposide, DAPI, and 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
(Sigma-Aldrich, St. Louis, MO, USA), penicillin G sodium
salt and streptomycin sulfate (Gibco, Grand Island, NY,
USA), and phosphate bufered saline (PBS) (Wako Pure
Chemical Industries, Osaka, Japan). All other chemicals
used were of biochemical reagent grade.
(20R)‑11α,12β,14β,20‑tetrahydroxy‑5α‑pregnan‑3β‑yl O‑
(6‑deoxy‑3‑O‑methyl‑β‑d‑allopyranosyl)‑(1 →4)‑O‑β‑d‑
oleandropyranoyl‑(1 →4)‑β‑d‑cymaropyranoside (1)
An amorphous solid; [α]2D5 −17.1 (c 0.10, MeOH); IR (flm)
νmax 3434 (OH), 2930 (CH) cm−1; HRESI-TOF-MS m/z
839.4759 [M+Na]+ (calcd for C42H72O15Na, 839.4769); 1H-
NMR (500 MHz, C5D5N) δ 4.15 (1H, m, H-20), 4.00 (1H,
dd, J=9.8, 9.8 Hz, H-11), 3.94 (1H, m, H-3), 3.50 (1H, d,
J=9.8 Hz, H-12), 1.72 (3H, s, Me-18), 1.49 (3H, d, J=6.5 Hz,
Me-21), 1.04 (3H, s, Me-19), signals for sugar moiety (see
Table 3); 13C-NMR (125 MHz, C5D5N) (see Tables 2, 3).
(20R)‑11α,12β,14β,20‑Tetrahydroxy‑5α‑pregnan‑3β‑yl O‑
(6‑deoxy‑3‑O‑methyl‑β‑d‑allopyranosyl)‑(1 →4)‑O‑β‑d‑
oleandropyranosyl‑(1 →4)‑β‑d‑cymaropyranoside (2)
Plant material
An amorphous solid; [α]25 − 21.2 (c 0.10, MeOH); IR
(film) νmax 3396 (OH), 29D24 (CH) cm−1; HRESI-TOF-
MS m/z 1001.5293 [M+Na]+ (calcd for C48H82O20Na,
The plant material defned as the bark of M. cundurango
was purchased from Tochimoto Tenkaido Co., Ltd (Osaka,
Japan) in May 2012. A voucher specimen has been deposited
in our laboratory (voucher no. Mc-2012-001, Department of
Medicinal Pharmacognosy).
1
1001.5297); H-NMR (500 MHz, C5D5N) δ 4.13 (1H, m,
H-20), 3.96 (1H, m, H-11), 3.91 (1H, m, H-3), 3.47 (1H, m,
H-12), 1.68 (3H, s, Me-18), 1.46 (3H, d, J=6.6 Hz, Me-21),
1.00 (3H, s, Me-19), signals for sugar moiety, (see Table 3);
13C-NMR (125 MHz, C5D5N) (see Tables 2, 3).
Extraction and isolation
(20R)‑11α,12β,14β,20‑Tetrahydroxy‑5α‑pregnan‑3β‑yl O‑
(6‑deoxy‑3‑O‑methyl‑β‑d‑allopyranosyl)‑(1 →4)‑O‑β‑d‑
oleandropyranosyl‑(1 →4)‑β‑d‑digitoxopyranoside (3)
The bark of M. cundurango (5.0 kg) was extracted twice
with hot MeOH (10 L). After removal of the solvent under
reduced pressure, the MeOH extract (500 g) was passed
through a Diaion HP-20 column, successively eluted with
H2O–MeOH (7:3) (6 L), H2O–MeOH (1:1) (5 L), MeOH
An amorphous solid; [α]D25 −3.2 (c 0.10, MeOH); IR (flm)
νmax 3410 (OH), 2929 (CH) cm−1; HRESI-TOF-MS m/z
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Table 2 13C-NMR chemical
shift assignments for the
aglycone moieties of 1–7 in
C5D5N
Position
1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
39.7
30.6
76.6
35.6
45.4
29.9
28.7
40.9
52.3
37.3
72.0
81.4
54.0
84.2
33.5
27.4
55.1
11.6
12.4
70.3
23.6
40.0
30.5
76.6
35.6
45.3
29.9
28.6
40.8
52.2
38.0
72.0
81.4
53.9
84.2
33.4
27.3
55.0
11.5
12.4
70.3
23.6
39.7
30.6
76.7
35.7
45.4
29.9
28.7
40.9
52.3
38.0
72.0
81.5
53.9
84.3
33.4
27.3
55.1
11.5
12.4
70.3
23.5
39.6
30.5
76.6
35.6
45.3
29.9
28.5
40.4
52.2
37.9
72.1
81.1
54.1
84.2
33.1
25.3
52.2
11.6
12.4
77.3
18.2
38.5
30.4
76.2
35.5
44.9
29.6
28.9
40.7
50.8
38.0
74.6
78.8
54.4
83.7
33.3
27.1
54.6
11.5
12.6
70.2
23.5
40.4
30.1
77.7
39.7
40.5
30.1
77.7
39.8
139.5
118.8
35.4
139.5
118.8
35.5
75.9
49.0
39.2
71.7
78.7
55.6
85.5
36.6
24.3
59.4
13.6
18.0
76.0
49.1
39.3
71.7
78.7
55.6
85.5
36.7
24.3
59.4
13.6
18.1
9
10
11
12
13
14
15
16
17
18
19
20
21
1′
213.2
31.4
170.1
21.5
213.3
31.5
170.1
21.5
2′
1″
2″
3″
4″
5″
6″
7″
167.0
120.2
144.4
135.0
128.4
129.1
130.4
167.2
118.0
146.5
134.6
128.8
129.3
130.9
167.2
118.1
146.5
134.7
128.8
129.4
131.0
825.4639 [M+Na]+ (calcd for C41H70O15Na, 825.4612); 1H-
NMR (500 MHz, C5D5N) δ 1.70 (3H, s, CH3-18), 1.48 (3H,
d, J = 6.4 Hz, CH3-21), 1.02 (3H, s, CH3-19), signals for
sugar moiety (see Table 3); 13C-NMR (125 MHz, C5D5N)
(see Tables 2, 3).
moiety (see Table 3); 13C-NMR (125 MHz, CD3OD) (see
Tables 2, 3).
(20R)‑11α‑[((E)‑Cinnamoyl)oxy]‑12β,14β,20‑trihydroxy‑
5α‑pregnan‑3β‑yl O‑(6‑deoxy‑3‑O‑methyl‑β‑d‑
allopyranosyl)‑(1 →4)‑O‑β‑d‑oleandropyranosyl‑
(1 →4)‑β‑d‑cymaropyranoside (5)
(20R)‑[20‑(β‑d‑Glucopyranosyl)oxy]‑11α,12β,14β‑
trihydroxy‑5α‑pregnan‑3β‑yl O‑(6‑deoxy‑3‑O‑methyl‑
β‑d‑allopyranosyl)‑(1 →4)‑O‑β‑d‑oleandropyranosyl‑
(1 →4)‑β‑d‑cymaropyranoside (4)
An amorphous solid; [α]2D5 15.8 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 276.5 (4.16) nm; IR (flm) νmax 3435
(OH), 2930 (CH), 1708 (C=O) cm−1; HRESI-TOF-MS m/z
969.5184 [M+Na]+ (calcd for C51H78O16Na, 969.5188); 1H-
NMR (500 MHz, C5D5N) δ 7.92 (1H, d, J=16.0 Hz, Cin),
7.50 (2H, m, Cin), 7.33 (1H, m, Cin), 7.32 (2H, m, Cin), 6.77
(1H, d, J=16.0 Hz, Cin), 5.81 (1H, t-like, J=9.7 Hz, H-11),
5.36 (1H, d, J=5.0 Hz, H-6), 3.71 (1H, d, J=9.7 Hz, H-12),
1.77 (3H, s, CH3-18), 1.46 (3H, d, J=6.4 Hz, CH3-21), 1.12
An amorphous solid; [α]D25 12.5 (c 0.10, MeOH); IR (flm)
νmax 3413 (OH), 2927 (CH) cm−1; HRESI-TOF-MS m/z
1001.5300 [M+Na]+ (calcd for C48H82O20Na, 1001.5297);
1H-NMR (500 MHz, C5D5N) δ 4.42 (1H, m, H-20), 3.92
(1H, dd, J = 9.3, 9.3 Hz, H-11), 3.89 (1H, m, H-3), 3.44
(1H, d, J=9.3 Hz, H-12), 1.68 (3H, s, Me-18), 1.53 (3H, d,
J=6.6 Hz, Me-21), 1.00 (3H, s, Me-19), signals for sugar
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1 3

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1 3

Journal of Natural Medicines
(3H, s, CH3-19), signals for sugar moiety (see Table 3); 13C-
NMR (125 MHz, C5D5N) (see Tables 2, 3).
(99:1:0.1) and CHCl3–MeOH–H2O (19:1:0.1) to yield
d-cymarose (0.4 mg from 1; 0.7 mg from 6; 0.5 mg from
7), d-digitoxose (0.9 mg from 3; 0.5 mg from 7), and O-(6-
deoxy-3-O-methyl-β-d-allopyranosyl)-(1 → 4)-d-olean-
dropyranose (1.4 mg from 1; 0.7 mg from 3; 0.7 mg from
7). The sugars were analyzed by HPLC under the follow-
ing conditions: column, Shodex sugar SC1011 (8.0 mm
i.d.×300 mm, 5 µm; Showa-denko, Tokyo, Japan); solvent,
H2O; fow rate, 1.0 mL/min; temperature, 80 °C; detection,
optical rotation (OR). The identifcation of d-cymarose and
d-digitoxose was achieved through a comparison of their
retention times (tR) and ORs with those of authenticated
standard samples; tR (min): d-cymarose (8.04, positive OR)
and d-digitoxose (9.32, positive OR). Identifcation of O-(6-
deoxy-3-O-methyl-β-d-allopyranosyl)-(1→4)-d-oleandro-
pyranose was achieved through a comparison of the specifc
rotation and the 1H and 13C NMR spectra with those of lit-
erature data.
11α‑Acetoxy‑12β‑[((E)‑cinnamoyl)oxy]‑8β,
14β‑dihydroxy‑20‑oxo‑pregn‑5‑en‑3β‑yl
O‑β‑d‑cymaropyranosyl‑(1 →4)‑β‑d‑cymaropyranoside (6)
An amorphous solid; [α]2D5 112.2 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 280.5 (4.31) nm; IR (flm) νmax 3429
(OH), 2932 (CH), 1744 and 1713 (C=O) cm−1; HRESI-
TOF-MS m/z 863.4201 [M+Na]+ (calcd for C46H64O14Na,
1
863.4194); H-NMR (500 MHz, C5D5N) δ 8.12 (1H, d,
J = 16.0 Hz, Cin), 7.62 (2H, m, Cin), 7.36 (1H, m, Cin),
7.35 (2H, m, Cin), 6.92 (1H, d, J=16.0 Hz, Cin), 6.34 (1H,
t-like, J=10.0 Hz, H-11), 5.50 (1H, d, J=10.0 Hz, H-12),
5.36 (1H, d, J = 5.0 Hz, H-6), 2.20 (3H, s, CH3-21), 1.96
(3H, s, OAc-11), 1.70 (3H, s, CH3-18), 1.61 (3H, s, CH3-19),
signals for sugar moiety (see Table 3); 13C-NMR (125 MHz,
C5D5N) (see Tables 2, 3).
O-(6-Deoxy-3-O-methyl-β-d-allopyranosyl)-(1→4)-β-d-
oleandropyranose: an amorphous solid; [α]2D5 7.4 (c 0.10,
1
11α‑Acetoxy‑12β‑[((E)‑cinnamoyl)oxy]‑8β,14β‑dihydroxy‑2
0‑oxo‑pregn‑5‑en‑3β‑yl O‑(6‑deoxy‑3‑O‑methyl‑β‑d‑
allopyranosyl)‑(1 →4)‑O‑β‑d‑oleandropyranosyl‑(1 →4)‑
O‑β‑d‑digitoxopyranosyl‑β‑d‑cymaropyranoside (7)
MeOH); H-NMR (500 MHz, CDCl3) δ 5.34 (1H, br s,
α-Ole-1), 4.82 (1H, d, J = 8.3 Hz, All-1), 4.81 (1H, dd,
J = 8.3, 1.7 Hz, β-Ole-1), 4.00 (1H, dq, J = 9.3, 6.3 Hz,
α-Ole-5), 3.80 (1H, dd, J=6.0, 2.8 Hz, All-3), 3.77 (1H, dd,
J=6.4, 4.6 Hz, α-Ole-3), 3.67 (3H, s, All-3-OMe), 3.56 (1H,
dq, J=9.7, 6.1 Hz, All-5), 3.49 (1H, m, All-2), 3.43 (1H, dd,
J=4.6, 2.8 Hz, β-Ole-3), 3.41 (1H, m, β-Ole-5), 3.40 (3H,
s, α-Ole-3-OMe), 3.40 (3H, s, β-Ole-3-OMe), 3.37 (1H, m,
α-Ole-4), 3.37 (1H, m, β-Ole-4), 3.20 (1H, m, All-4), 2.45
(1H, ddd, J=13.3, 4.6, 1.8 Hz, β-Ole-2eq), 2.31 (1H, ddd,
J=12.8, 4.6, 1.8 Hz, α-Ole-2eq), 1.55 (1H, m, α-Ole-2ax),
1.43 (1H, m, β-Ole-2ax), 1.38 (3H, d, J=6.0 Hz, β-Ole-6),
1.32 (3H, d, J=6.3 Hz, α-Ole-6), 1.27 (3H, d, J=6.1 Hz,
All-6); 13C-NMR (125 MHz, CDCl3) δ 91.9, 34.6, 76.1,
80.1, 67.6, 18.7 (α-Ole-1–C-6), 56.3 (α-Ole-3-OMe), 94.0,
37.1, 78.9, 80.1, 79.4, 18.9 (β-Ole-1–6), 56.3 (β-Ole-3-
OMe), 99.1 72.3, 81.2, 73.0, 71.6, 18.1 (All-1–C-6), 62.2
(All-3-OMe).
An amorphous solid; [α]2D5 64.4 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 280.0 (4.19) nm; IR (flm) νmax 3446
(OH), 2931 (CH), 1746, 1713 and 1698 (C=O) cm−1
;
HRESI-TOF-MS m/z 1153.5536 [M+Na]+ (calcd for
1
C59H86O21Na, 1153.5559); H-NMR (500 MHz, C5D5N)
δ 8.12 (1H, d, J = 16.0 Hz, Cin), 7.63 (2H, m, Cin), 7.36
(1H, m, Cin), 7.35 (2H, m, Cin), 6.92 (1H, d, J=16.0 Hz,
Cin), 6.35 (1H, t-like, J = 10.1 Hz, H-11), 5.51 (1H, d,
J = 10.1 Hz, H-12), 5.36 (1H, br d, J = 5.4 Hz, H-6), 2.20
(3H, s, CH3-21), 1.98 (3H, s, OAc-11), 1.70 (3H, s, CH3-18),
1.62 (3H, s, CH3-19), signals for sugar moiety (see Table 3);
13C-NMR (125 MHz, C5D5N) (see Tables 2, 3).
Acid hydrolysis of 1, 3, 6, and 7
Enzymatic hydrolysis of 2
Compounds 1 (5.7 mg), 3 (5.1 mg), 6 (3.2 mg), and 7
(4.1 mg) were dissolved separately in 0.025 M HCl (diox-
ane–H2O, 1:1, 2 mL). Each solution was heated at 95 °C for
1.5 h under an argon atmosphere. After cooling, the reaction
mixture was neutralized by passing through an Amberlite
IRA-96SB column and chromatographed on silica gel eluted
with CHCl3–MeOH (19:1) to yield the aglycone and sugar
fractions. The aglycone fraction was subjected to a silica gel
column chromatography eluted with CHCl3–MeOH–H2O
(19:1:0.1) to yield 1a (1.6 mg from 1; 2.4 mg from 3). The
aglycones of 6 and 7 were decomposed under acidic con-
ditions and could not be obtained. The sugar fraction was
subjected to silica gel CC eluted with EtOAc–MeOH–H2O
Compound 2 (6.0 mg) was treated with β-d-glucosidase
(EC 232-589-7; Sigma-Aldrich; 62.0 mg) in NaOAc/KOAc
bufer (pH 5.0, 5 mL) at room temperature for 168 h. The
reaction mixture was chromatographed on silica gel eluted
with CHCl3–MeOH–H2O (7:4:1) to yield 1 (0.3 mg) and a
sugar fraction (0.3 mg). The sugar fraction was analyzed
by HPLC under the following conditions: column, Capcell
Pak NH2 UG80 (4.6 mm i.d. × 250 mm, 5 µm, Shiseido
Co. Ltd., Tokyo, Japan); solvent, MeCN-H2O (17:3); fow
rate, 1.0 mL/min; detector, OR. The identifcation of d-glu-
cose present in the sugar fraction was achieved through a
1 3

Journal of Natural Medicines
comparison of its retention time and OR with those of an
6 h, and the cells were collected by centrifugation at 500 g
for 5 min. The cell pellets were resuspended in 250 µL of
MitoCapture™ solution, incubated at 37 °C for 15 min, and
then centrifuged again for 5 min. The collected pellets were
resuspended in 200 µL of incubation bufer and observed
under a fuorescence microscope.
authentic sample; tR (min): d-glucose (11.79, positive OR).
Cell culture and assay for cytotoxic activity
against HL‑60, A549, and TIG‑3 cells
The cells were cultured in RPMI 1640 medium (HL-60
cells) or MEM (A549 and TIG-3 cells) containing heat-
inactivated 10% (v/v) FBS supplemented with l-glutamine,
100 U/mL penicillin G sodium salt, and 100 µg/mL strep-
tomycin sulfate in a humidifed incubator at 37 °C with an
atmosphere of 5% CO2. The cells (HL-60 cells: 4×104 cells/
mL; A549 cells: 1×104 cells/mL; TIG-3 cells: 2×104 cells/
mL) were treated with each compound for 72 h continuously,
followed by the measurement of cell viability using an MTT
assay. Dose–response curves were plotted for compounds 1,
5–9, 11, and 12, and the concentrations required to obtain
50% cell growth inhibition (IC50) were calculated.
Statistical analysis
Statistical analysis of the results was performed by one-way
analysis of variance followed by Dunnett’s test. A probabil-
ity (P) value of < 0.001 was considered to represent a sta-
tistically signifcant diference.
Acknowledgements This work was fnancially supported in part by
the Japan Society for the Promotion of Sciences (JSPS) KAKENHI
(Grant number 26860069).
DAPI staining
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DAPI (0.5 µg/mL in PBS) at room temperature. The stained
cells were then observed immediately under a fuorescence
microscope.
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