Cycloartane glycosides from Astragalus aureus

Article abstract of DOI:10.1016/j.phytochem.2011.02.006

Eight cycloartane-type triterpene glycosides (1-8) were isolated from Astragalus aureus Willd along with ten known cycloartane-type glycosides (9-18). Their structures were established by the extensive use of 1D and 2D-NMR experiments along with ESIMS and HRMS analyses. Compounds 1-5 are cyclocanthogenin glycosides, whereas compounds 6-8 are based on cyclocephalogenin as aglycon, more unusual in the plant kingdom, so far reported only from Astragalus spp. Moreover, for the first time monoglycosides of cyclocanthogenin (5) and cyclocephalogenin (7, 8) are reported. All of the compounds tested for their cytotoxic activities against a number of cancer cell lines. Among the compounds, only 8 exhibited activity versus human breast cancer (MCF7) at 45 μM concentration.

Full text of DOI:10.1016/j.phytochem.2011.02.006

Phytochemistry 72 (2011) 761–768
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Cycloartane glycosides from Astragalus aureus
a
a,
⇑
b
c
b
Derya Gülcemal , Özgen Alanku ßs -Çalı ßs kan , Angela Perrone , Fevzi Özgökçe , Sonia Piacente ,
d,
⇑
Erdal Bedir
a
_
Department of Chemistry, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey
Department of Pharmaceutical Sciences, Salerno University, 84084 Fisciano (Salerno), Italy
Department of Biology, Faculty of Science and Letters, Yüzüncü Yıl University, 65180 Van, Turkey
b
c
d
_
Department of Bioengineering, Faculty of Engineering, Ege University, 35100 Bornova-Izmir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight cycloartane-type triterpene glycosides (1–8) were isolated from Astragalus aureus Willd along with
ten known cycloartane-type glycosides (9–18). Their structures were established by the extensive use of
Received 21 October 2010
Received in revised form 4 February 2011
Available online 4 March 2011
1D and 2D-NMR experiments along with ESIMS and HRMS analyses. Compounds 1–5 are cyclocanthog-
enin glycosides, whereas compounds 6–8 are based on cyclocephalogenin as aglycon, more unusual in
the plant kingdom, so far reported only from Astragalus spp. Moreover, for the first time monoglycosides
of cyclocanthogenin (5) and cyclocephalogenin (7, 8) are reported. All of the compounds tested for their
cytotoxic activities against a number of cancer cell lines. Among the compounds, only 8 exhibited activity
_
Dedicated to Prof. Ihsan Çalı ßs on the
occasion of his 60th birthday
versus human breast cancer (MCF7) at 45 lM concentration.
Keywords:
Astragalus aureus
Saponin
Ó 2011 Elsevier Ltd. All rights reserved.
Cycloartane
Cyclocephalogenin
Cyclocanthogenin
Cytotoxic activity
1
. Introduction
Astragalus species (Bedir et al., 1998a,b, 1999; Polat et al., 2009,
2
010; Horo et al., 2010).
The genus Astragalus belonging to the Leguminosae family is
Cycloartane- and oleanane-type glycosides from Astragalus spe-
widely distributed throughout the temperate regions of the world,
located principally in Europe, Asia and North America. About 2000
species have been described, 380 of which are represented in the
flora of Turkey (Ríos and Waterman, 1997; Davis 1970). The roots
of various Astragalus species represent very old and well-known
drugs in traditional medicine for the treatment of nephritis, diabe-
tes, uterine cancer and as antiperspirant, diuretic, and tonic (Tang
and Eisenbrand, 1992). In Turkish folk medicine, the aqueous ex-
tracts of some Astragalus species (declared by the healer) are used
to treat leukemia as well as for wound healing (Çalı ßs et al., 1997;
Bedir et al., 2000a).
Besides polysaccharides, saponins are the major class of biolog-
ically active compounds from Astragalus species thoroughly inves-
tigated (Tang and Eisenbrand, 1992). Chemical studies on
Astragalus saponins have resulted in isolation of cycloartane- and
oleanane-type glycosides (Ríos and Waterman, 1997; Verotta and
El-Sebakhy, 2001). A series of cycloartane-type triterpenoidal sap-
onins were isolated from our earlier investigations on Turkish
cies have shown interesting biological properties, including immu-
nostimulating (Çalı ßs et al., 1997; Bedir et al., 2000a; Yesilada et al.,
2005), anti-protozoal (Özipek et al., 2005), antiviral (Gariboldi
et al., 1995), and cytotoxic activities (Tian et al., 2005). On the basis
of interesting activities reported for many Astragalus species and
for cycloartane glycosides, and as a part of our ongoing research
of new bioactive compounds from Turkish Astragalus species, we
carried out a study on Astragalus aureus Willd (Leguminosae). This
paper reports the isolation of eight new cycloartane-type triter-
pene glycosides (1–8) from the methanol extract of A. aureus along
with ten known cycloartane-type glycosides (9–18). Their struc-
tures were elucidated by extensive spectroscopic methods includ-
1
13
ing 1D ( H, C and TOCSY) and 2D-NMR (DQF-COSY, HSQC, HMBC
and ROESY) experiments as well as ESIMS and HRMS analysis.
Additionally, all of the compounds were screened for their antipro-
liferative activities versus a number of cancer cell lines.
2. Results and discussion
The HRMALDITOF mass spectrum of 1 (m/z 1057.5563 [M+Na]+,
⇑
Corresponding authors. Tel./fax: +90 232 3888264 (Ö. Alanku sß -Çalı ßs kan), tel./
calcd for C51
of C51 21. The ESIMS spectrum showed a major ion peak at m/z
1057.65 which was assigned to [M+Na] . The MS/MS of this ion
86
H O21Na, 1057.5559) supported a molecular formula
fax: +90 232 388 4955 (E. Bedir).
86
H O
E-mail addresses: ozgen.alankus.caliska@ege.edu.tr (Ö. Alanku sß -Çalı ßs kan), erdal.
bedir@ege.edu.tr (E. Bedir).
+
0
031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.02.006

7
62
D. Gülcemal et al. / Phytochemistry 72 (2011) 761–768
+
showed a peak at m/z 907.53 [M+Na-150] , corresponding to the
tertiary methyl groups at d 1.28 (3H, s), 1.20 (3H, s), 1.18 (3H, s),
1.17 (3H, s), 1.03 (3H, s) and 1.01 (3H, s), a secondary methyl group
at d 0.98 (d, J = 6.5 Hz), and four methine proton signals at d 4.48
(ddd, J = 8.0, 8.0, 5.2 Hz), 3.54 (ddd, J = 9.5, 9.5, 4.5 Hz), 3.41 (dd,
J = 10.5, 2.4 Hz) and 3.23 (dd, J = 11.3, 4.0 Hz), which were indica-
tive of secondary alcoholic functions. The NMR data of the aglycon
moiety of 1 were in good agreement with those reported for cyclo-
canthogenin with glycosidation shifts for C-3 (d 89.6) and C-6 (d
3
loss of a pentose unit. In the MS spectrum peaks at m/z 761.46
M+Na-150–146] , corresponding to the loss of a deoxy–hexose
unit, and 629.42 [M+Na-150–146–132] , due to the loss of a pen-
tose unit, were observed. A detailed comparison of the NMR data
H, C, HSQC, HMBC, COSY) of compounds 1–5 (Fig. 1) showed
that the aglycon moiety was identical in all five compounds. In par-
ticular, the H NMR spectrum of 1 showed signals due to a cyclo-
propane methylene at d 0.59 and 0.25 (each 1H, d, J = 4.2 Hz), six
+
[
+
1
13
(
1
1
3
79.0) (Isaev et al., 1992). The C-NMR chemical shift for C-24
21
OH
1
8
2
0
26
24
1
6
7
12
19
1
OH
OR
3
1
4
2
7
1
9
3
7
30
5
R O
1
28
29
OR2
O
O
HO
HO
OH
HO
1
R
1
=
R
2
=
HO
R = H
3
β-
D-xyl I
OH
D-xyl II
β-
O
O
O
α-
L
-ara
HO
O
HO
α-
OH
L-rha
HO
O
O
HO
HO
2
3
4
5
R = HO
1
R =
HO
R = H
3
2
OH
OH
D-xyl II
β-
D
-xyl I
β-
OH
O
O
O
HO
HO
HO
HO
R = HO
1
R
R
2
2
=
=
HO
R
R
3
3
=
=
OH
OH
OH
-glc
β-
D
-xyl I
β-
D
-xyl II
β-D
OH
OH
O
O
O
HO
HO
HO
β-
HO
HO
β-
R = HO
1
OH
D
-glc I OH
-glc II OH
D
β-
D
-xyl
OH
O
HO
HO
R = H
R
2
=
R = H
3
1
OH
β-D-glc
OH
21
24
25
2
6
2
0
O
2
7
OH
R O
1
OR2
O
6
R = HO
R = H
1
2
HO
OH
HO
R = H
β-
D-xyl
O
O
α-
L
-ara
OH
OH
O
HO
HO
7
8
1
R =
2
OH
-glc
β-D
O
HO
R =
HO
2
R = H
1
OH
-xyl
β-D
Fig. 1. Structures of compounds 1–8.

D. Gülcemal et al. / Phytochemistry 72 (2011) 761–768
763
can be regarded as a characteristic parameter in the determination
of the configurations of C-24. In the case of the 24R configuration,
the chemical shift for C-24 gives resonance at 79.9–80.6 ppm
reported for the
For the
stant has been reported not to be diagnostic on its own, owing to
a
anomer of rhamnopyranose (Kasai et al., 1979).
L
-arabinopyranosyl unit the value of JH1-H2 coupling con-
4
(Isaev et al., 1983, 1982; Agzamova and Isaev; 1998), while C-24
1
the high conformational mobility of arabinopyranosides ( C ?
1
gives resonance at 77.0–78.2 ppm for the 24S configuration (Isaev
4
C ) (Breitmaier and Voelter, 1987). Evidence supporting an a-L-
et al., 1992; Bedir et al., 2000b; Hirotani et al., 1994a,b; Fadeev
arabinopyranoside configuration in rapid conformational exchange
1
3
et al., 1988). The C NMR data of compound 1 are comparable to
those reported for analogous compounds having a 24S configura-
tion. The relative configurations of the oxygenated carbons were
determined by the multiplicity of the vicinal proton–proton cou-
pling constants to be b-OH for C-3 (d 3.23, dd, J = 11.3, 4.0 Hz.
was obtained from ROESY experiments. NOe’s were observed from
H
ara-1 to Hara-2 and Hara-1 to Hara-3 as expected for 1 and 4
C C
4 1
conformations respectively. The nOe Hara-1-Hara-3 would not be
1
4
expected for both C
4
- C
1
-b-
L-arabinopyranosides. An nOe was also
observed between Hara-1 and Hara-5 as expected for an
pyranoside in a C conformation.
1
a-L
-arabino-
4
Hax-3),
a-OH for C-6 (d 3.54, ddd, J = 9.5, 9.5, 4.5 Hz, Hax-6) and
b-OH for C-16 (d 4.48, ddd, J = 8.0, 8.0, 5.2 Hz, Hax-16). Additionally,
the relative configuration of C-20 was derived by the ROESY spec-
Glycosidation shifts were observed for C-2xyl I (d 79.9) and C-
2ara (d 75.9). An unambiguous determination of the sequence and
trum that showed key correlations between Me-30
(d 4.48) and H-17 (d 1.74) signals, and between H-17
Me-21 (d 0.98) signals.
a
(d 1.01), H-
linkage sites was obtained from the HMBC spectrum, which
showed key correlation peaks between the proton signal at d
4.49 (H-1xyl ) and the carbon resonance at d 89.6 (C-3), d 4.98
I
1
6a
a
a
and
a
For compound 1, a secondary methyl group at d 1.31 (d, J =
.5 Hz) along with four anomeric protons at d 5.06 (d, J = 1.2 Hz),
.98 (d, J = 3.7 Hz), 4.49 (d, J = 7.5 Hz) and 4.33 (d, J = 7.5 Hz) were
(H-1ara) and d 79.9 (C-2xyl), d 5.06 (H-1rha) and d 75.9 (C-2ara),
and the proton signal at d 4.33 (H-1xyl II) and the carbon resonance
at d 79.0 (C-6). The D configuration of xylose unit and the L config-
uration of arabinose and rhamnose units were established after
hydrolysis of 1 with 1 N HCl, trimethylsilation and determination
of the retention times by GC (De Marino et al., 2003).
6
4
additionally observed. The chemical shifts of all the individual pro-
tons of the four sugar units were ascertained from a combination of
1
D-TOCSY and DQF-COSY spectral analyses, and the 13C chemical
shifts of their relative attached carbons were assigned unambigu-
ously from the HSQC spectrum (Table 1). These data showed the
On the basis of all these evidence, the structure of the new com-
pound 1 was established as 3-O-[
arabinopyranosyl-(1?2)-b- -xylopyranosyl]-6-O-b-
syl-3b,6 ,16b,24(S),25-pentahydroxycycloartane.
The molecular formula of compound 2 was established as
a
-L
-rhamnopyranosyl-(1?2)-
a-L-
presence of one
anosyl units (d 4.49 and 4.33) and one
.98). The configuration of the rhamnose unit was deduced from
a-rhamnopyranosyl unit (d 5.06), two b-xylopyr-
D
D-xylopyrano-
a-arabinopyranosyl unit (d
a
4
a
+
the H-1/C-1 J value = 169 Hz, measured from the residual direct
correlation observed in the HMBC spectrum, in agreement with that
40 68
C H O13 by HRMALDITOFMS analysis (m/z 779.4561 [M + Na] ,
1
calcd for C40H
68
O
13Na, 779.4558). The H NMR spectrum of 2
Table 1
1
H NMR data (J in Hz) of the sugar portions of compounds 1–5 (600 MHz, d ppm, in CD
3
OD).
1
2
3
4
5a
d
C
d
H
(J in Hz)
-Xyl I (at C-3)
105.9 4.49, d (7.5)
d
C
d
H
(J in Hz)
-Xyl I (at C-3)
106.9 4.31, d (7.5)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
-Xyl I (at C-3)
106.8 4.31, d (7.5)
d
C
d
H
(J in Hz)
-Glc (at C-6)
104.6 4.36, d (7.5)
b-
D
b-
D
b-
D
-Xyl I (at C-3)
b-
D
b-D
1
2
3
4
5
107.0 4.31, d (7.5)
79.9
77.0
71.0
66.2
3.42, dd (9.2, 7.5)
3.54, t (9.2)
3.54, m
75.1
77.7
71.0
3.22, dd (9.2, 7.5)
3.33, t (9.2)
3.50, m
3.86, dd (11.7, 5.2) 66.3
3.20, t (11.7)
75.1
77.6
70.9
3.22, dd (9.2, 7.5)
3.32, t (9.2)
3.49, m
3.86, dd (11.7, 5.2)
3.20, t (11.7)
75.0
78.0
71.0
66.3
3.22, dd (9.2, 7.5)
3.34, t (9.2)
3.50, m
3.85, dd (11.7, 5.2)
3.21, t (11.7)
75.4
78.2
71.3
77.4
3.21, dd (7.5, 9.0)
3.36, t (9.0)
3.31, t (9.0)
3.28, ddd (3.5, 4.5, 9.0)
3.90, dd (11.7, 5.2) 66.5
.23, t (11.7)
3
6
62.7
3.88, dd (3.5, 12.0)
3.69, dd (4.5, 12.0)
a
-
L-Ara (at C-2xyl
)
b-
D
-Xyl II (at C-6)
b-
D-Xyl II (at C-6)
b-D-Glc I (at C-6)
1
2
3
4
5
101.8 4.98, d (3.7)
105.1 4.32, d (7.5)
105.1 4.33, d (7.5)
104.6 4.36, d (7.5)
75.9
72.1
67.9
63.2
3.90, dd (8.5, 3.7)
3.84, dd (8.5, 3.0)
3.84, m
3.97, dd (11.9, 2.0) 66.5
.48, dd (11.9, 3.0)
75.1
77.7
71.0
3.22, dd (9.2, 7.5)
3.33, t (9.2)
3.50, m
75.1
77.6
70.9
3.20, dd (9.2, 7.5)
3.32, t (9.2)
3.49, m
75.0
78.2
71.3
77.2
3.20, dd (7.5, 9.0)
3.36, t (9.0)
3.30, t (9.0)
3.31, ddd (3.5, 4.5, 9.0)
3.86, dd (11.7, 5.2) 66.3
3.20, t (11.7)
3.86, dd (11.7, 5.2)
3.20, t (11.7)
3
6
62.7
3.86, dd (3.5, 12.0)
3.68, dd (4.5, 12.0)
a
-
L-Rha (C-2ara
)
b-
D-Glc (at C-25)
b-D-Glc II (at C-25)
1
2
3
4
5
6
101.8 5.06, d (1.2)
97.6
75.0
77.9
71.3
77.5
62.4
4.55, d (7.5)
3.19, dd (7.5, 9.0)
3.40, t (9.0)
3.31, t (9.0)
3.31, ddd (3.5, 4.5, 9.0) 77.4
97.5
75.0
77.9
71.3
4.55, d (7.5)
3.19, dd (7.5, 9.0)
3.41, t (9.0)
72.1
72.1
73.8
70.1
17.9
3.90, dd (1.2, 3.2)
3.72, dd (3.2, 9.3)
3.43, t (9.3)
3.89, m
3.33, t (9.0)
3.28, ddd (3.5, 4.5, 9.0)
3.86, dd (3.5, 12.0)
3.68, dd (4.5, 12.0)
1.31, d (6.5)
3.86, dd (3.5, 12.0)
3.68, dd (4.5, 12.0)
62.7
3
.68, dd (4.5, 12.0)
-Xyl II (at C-6)
105.2 4.33, d (7.5)
b-
D
1
2
3
4
5
75.4
77.7
71.0
66.3
3.22, dd (9.2, 7.5)
3.34, t (9.2)
3.50, m
3.86, dd (11.7, 5.2)
3.20, t (11.7)
a
The chemical shift values of the sugar portion of 7 were superimposable with those reported for 5.

7
64
D. Gülcemal et al. / Phytochemistry 72 (2011) 761–768
showed signals for two anomeric protons at d 4.31 (d, J = 7.5 Hz)
and 4.32 (d, J = 7.5 Hz). The HSQC, HMBC, DQF-COSY and 1D-TOCSY
data led to identify these sugar units as two b-xylopyranosyl units.
The HMBC correlations between the proton signal at d 4.31 (H-1xyl
determined as reported for compound 1. The a orientation of the
–OH group at C-24 was determined by the ROE correlation be-
tween Me-27 (d 1.32) and H-24b (d 3.51) signals.
1
For the sugar region in the H NMR spectrum, two anomeric
I
) and the carbon resonance at d 89.6 (C-3) and the proton signal at
d 4.32 (H-1xyl II) and the carbon resonance at d 79.0 (C-6) allowed
us to determine the linkage site of the sugar units. Thus, the new
protons at d 4.49 (d, J = 3.7 Hz) and 4.48 (d, J = 7.5 Hz) were ob-
1
13
served. Complete assignments of the H and C NMR signals of
the sugar portion were accomplished by 1D-TOCSY, HSQC and
DQF-COSY experiments which led to the identification of one
compound
2
was elucidated as 3,6-di-O-b-
D-xylopyranosyl-
3
b,6 ,16b,24(S),25-pentahydroxycycloartane.
The HRMALDITOF mass spectrum of 3 (m/z 941.5090 [M + Na] ,
a
b-xylopyranosyl unit (d 4.48) and one a-arabinopyranosyl unit (d
+
4.49). The determination of the sequence and linkage sites was ob-
tained from the HMBC correlations which showed key correlation
peaks between the proton signals at d 4.48 (H-1xyl) and the carbon
calcd for C46
18. The H NMR spectrum of 3 displayed, in addition to sig-
nals of the aglycon moiety, signals for three anomeric protons at d
.55 (d, J = 7.5 Hz), 4.33 (d, J = 7.5 Hz) and 4.31 (d, J = 7.5 Hz). On
78
H O18Na, 941.5086) supported a molecular formula of
1
46 78
C H O
resonance at d 89.3 (C-3), and the proton signal at d 4.49 (H-1ara
and the carbon resonance at d 82.9 (C-2xyl). Thus, compound 6
was elucidated as 3-O-[ -arabinopyranosyl-(1?2)-b- -xylopyr-
anosyl]-3b,6 ,16b,24 -tetrahydroxy-20(R),25-epoxycycloartane.
The molecular formulas of compounds 7 and 8 were established
)
4
the basis of HSQC, HMBC, DQF-COSY and 1D-TOCSY correlations
two b-xylopyranosyl units (d 4.33 and 4.31) and one b-glucopyran-
osyl (d 4.55) were identified. Glycosidation shifts for the aglycon
moiety were observed for C-3 (d 89.6), C-6 (d 79.0) and C-25 (d
a-
L
D
a
a
as C36
H
60
O
10 and C35
H
58
O
9
by HRMALDITOFMS analysis (m/z
10Na, 675.4084, and m/z
58 9
645.3979 [M + Na] , calcd for C35H O Na, 645.3983), respectively.
+
8
1.2). Key correlation peaks in the HMBC spectrum were observed
60
675.4089 [M + Na] , calcd for C36H O
+
between the proton signal at d 4.31 (H-1xyl I) and the carbon reso-
nance at d 89.6 (C-3), d 4.33 (H-1xyl II) and d 79.0 (C-6), and be-
tween the proton signal at d 4.55 (H-1glc) and the carbon
resonance at d 81.2 (C-25). The configuration of glucose and
xylose was determined as D via hydrolysis followed by GC analysis.
The comparison of the NMR data of compounds 7 and 8 showed
that the two compounds differed only for the sugar unit attached
at C-6. The combination of 1D-TOCSY, HSQC and DQF-COSY data
led to determine the sugar units as b-glucopyranosyl (d 4.36, d,
J = 7.5 Hz) for 7 and b-xylopyranosyl (d 4.33, d, J = 7.5 Hz) for 8.
Therefore, the structures of compounds 7 and 8 were estab-
Therefore, the structure of 3 was established as 3,6-di-O-b-
pyranosyl-25-O-b- -glucopyranosyl-3b,6 ,16b,24(S),25-pentahydr-
D-xylo-
D
a
oxycycloartane accommodating a very rare chemical feature in
nature, a tridesmosidic arrangement.
lished as 6-O-b-
20(R),25-epoxycycloartane and 6-O-b-
24 -tetrahydroxy-20(R),25-epoxycycloartane, respectively.
Compounds 1–5 were based on cyclocanthogenin, one of the
most common aglycons in Astragalus genus, whereas compounds
6–8 were cyclocephalogenin glycosides, more unusual in the plant
kingdom, so far reported only from Astragalus spp. (Bedir et al.,
1998a; Sukhina et al., 2007; Agzamova and Isaev, 1999; Semmar
et al., 2001). Particularly, monoglycosides of cyclocanthogenin (5)
and cyclocephalogenin (7, 8) are reported for the first time.
D
-glucopyranosyl-3b,6
a
,16b,24
a
-tetrahydroxy-
D-xylopyranosyl-3b,6
a,16b,
The molecular formula of 4 was established as C47
H
80
O
19 by
a
+
HRMALDITOFMS analysis (m/z 971.5197 [M + Na] , calcd for
19Na, 971.5192). The comparison of the NMR data of com-
C
47
80
H O
pound 4 with those of compound 3 allowed us to determine that
the two compounds differed only by the presence of a b-glucopyr-
anosyl unit at C-6 in 4 instead of the b-xylopyranosyl unit in 3.
Thus, compound 4 was elucidated as 3-O-b-
,25-di-O-b- -glucopyranosyl-3b,6 ,16b,24(S),25-pentahydroxy-
cycloartane revealing another tridesmosidic glycoside.
The HRMALDITOF mass spectrum of 5 (m/z 677.4246 [M + Na] ,
calcd for C36 10Na, 677.4241) supported a molecular formula of
10. The H NMR spectrum of 5 displayed, in the sugar re-
D-xylopyranosyl-
6
D
a
Additionally, ten known cycloartane-type glycosides, 3-O-[
-rhamnopyranosyl-(1?2)-O- -arabinopyranosyl-(1?2)-O-b-
xylopyranosyl]-6-O-b- -glucopyranosyl-3b,6 ,16b,24(S),25-penta-
a-
+
L
a
-
L
D-
H
62
O
D
a
1
C
36
H
62
O
hydroxycycloartane (9) (Horo et al., 2010), oleifolioside B (10)
gion, only one anomeric proton signal at d 4.36 (d, J = 7.5 Hz). On
the basis of 1D-TOCSY, HSQC and DQF-COSY data this sugar unit
was identified as b-glucopyranosyl unit. The HMBC spectrum
showed the correlation between the proton signal at d 4.36 (H-
(Özipek et al., 2005), cyclocanthoside G (11) (Isaev et al., 1992),
cyclocanthoside E (12) (Isaev et al., 1992), 3-O-[
anosyl-(1?2)-O- -arabinopyranosyl-(1?2)-O-b-
syl]-6-O-b- -glucopyranosyl-3b,6 ,16b,24 -tetrahydroxy-20(R),
25-epoxycycloartane (13) (Horo et al., 2010), 3-O-[ -arabinopyr-
anosyl-(1?2)-O-b- -xylopyranosyl]-6-O-b- -glucopyranosyl-3b,
,16b,24 -tetrahydroxy-20(R),25-epoxycycloartane (14) (Horo
a-
L-rhamnopyr-
a
-
L
D-xylopyrano-
D
a
a
1glc) and the carbon resonance at d 80.1 (C-6), allowing us to eluci-
a-L
date compound 5 as 6-O-b-
D
-glucopyranosyl-3b,6
a
,16b,24(S),25-
D
D
pentahydroxycycloartane.
6a
a
The molecular formula of 6 was established as C40
H
66
O
13 by
et al., 2010), cyclocanthoside F (15), (Agzamova and Isaev, 1999),
cyclocephaloside I (16) (Bedir et al., 1998a), cyclotrisectoside (17)
(Sukhina et al., 2007) and macrophyllosaponin B (18) (Çalı ßs et al.,
1996) were isolated.
The antiproliferative activity of cycloartane glycosides has been
reported against several cancer lines including solid tumor
(HepG2), blood tumor (HL-60) and drug resistant tumor (R-HepG2)
(Kikuchi et al., 2007). In particular 16b,23:23,26:24,25-triepoxy-,
+
HRMALDITOFMS analysis (m/z 777.4405 [M + Na] , calcd for
13Na, 777.4401). The ESIMS mass spectrum showed the
40 66
C H O
+
major ion peak at m/z 777.74 which was assigned to [M + Na] . In
the MS/MS spectrum peaks at m/z 645.36 [M + Na-132] , corre-
sponding to the loss of a pentose unit, 495.41 [M + Na-150] , corre-
+
+
sponding to the loss of a further pentose unit, were observed. It
1
13
was apparent from their H and C NMR data that 6–8 possessed
1
the same aglycon moiety. In particular, the H NMR spectrum of 6
16b,23:16a,24-diepoxy- and 16b,23;22b,25-diepoxycycloartane
showed signals due to a cyclopropane methylene at d 0.57 and 0.43
derivatives have been reported for their cytotoxic activities (Nian
et al., 2010; Watanabe et al., 2002). Moreover, cancer chemopre-
ventive effects of natural and semisynthetic cycloartane-type and
related triterpenoids have been reported (Tian et al., 2005). Leish-
manicidal (Özipek et al., 2005) and immunomodulatory (Çalis
et al., 1997) activities have been reported for cyclocanthogenin
and cycloastragenol derivatives.
(
each 1H, d, J = 4.2 Hz), seven tertiary methyl groups at d 1.54 (3H,
s), 1.49 (3H, s), 1.32 (6H, s), 1.22 (3H, s), 1.05 (3H, s) and 0.97 (3H,
s) and four methine proton signals at d 4.64 (ddd, J = 8.0, 8.0,
5
.2 Hz), 3.47 (ddd, J = 9.5, 9.5, 4.5 Hz), 3.51 (br s) and 3.22 (dd,
J = 11.3, 4.0 Hz). The NMR data of compound 6 were in agreement
with those reported for 3b,6 ,16b,24 -tetrahydroxy-20(R),25-
a
a
epoxycycloartane named cyclocephalogenin, previously reported
from Astragalus spp. (Bedir et al., 1998b). The relative configura-
tions of the oxygenated carbons C-3, C-6, C-16 and C-20 were
On the basis of the biological activities reported for cycloartane
glycosides, the antiproliferative activity of compounds 1–18 was
tested in several cancer cell lines including HL-60 (human promy-

D. Gülcemal et al. / Phytochemistry 72 (2011) 761–768
765
elocytic leukemia), MCF-7 (human breast cancer), HT-29 (human
3 2
tography. Elution was carried out with CHCl –MeOH–H O
colon carcinoma), A549 (human lung adenocarcinoma), PC3 (hu-
(90:10:1, 500 ml) to give 8 (10.4 mg). Subfraction 8 (70 mg) was
man prostate cancer). Compounds 1–18 were tested in a range of
fractionated over an open column chromatography using silica
concentrations between 1 and 50
showed a weak activity. Compound 17 exhibited an IC50 value of
M against MCF7 cells, while compound 8 exhibited an IC50 va-
lue of 45 M against HL-60 cells. Compounds 12 and 14 showed an
IC50 value of 50 M against A549 and PC3 cells. All the other com-
pounds showed no activity (data not shown). Etoposide used as po-
sitive control showed IC50 values ranging from 0.5 M (HL-60 cells)
to 16 M (A549 cells).
l
M, but only few compounds
gel (30 g). Elution was performed with CHCl
tures (90:10:1, 500 ml) to afford (8.4 mg). Subfraction
(100 mg) was submitted to silica gel (30 g) column chromatogra-
phy with the solvent system CHCl –MeOH–H (90:10:1,
750 ml) to give 5 (5 mg). Subfraction 19 (100 mg) was subjected
to silica gel (30 g) column chromatography with the solvent sys-
3 2
–MeOH–H O mix-
6
9
30 l
l
3
2
O
l
l
tem CHCl
fraction 17 (100 mg) was applied to a reversed phase column
Lichroprep RP-18, 25–40 m, 30 g) using MeOH–H (6:4,
3 2
–MeOH–H O (90:10:1, 750 ml) to yield 14 (27 mg). Sub-
l
(
l
2
O
3
. Experimental
.1. General
Optical rotations were measured on a JASCO DIP 1000 polarim-
500 ml) to give 12 (7.3 mg). Subfraction 21 (118 mg) was chro-
matographed on a reversed phase material (Lichroprep RP-18,
3
25–40
49 mg) and 10 (8 mg). Subfraction 22 (140 mg) was purified on
a reversed phase column (Lichroprep RP-18, 25–40 m, 30 g) and
eluted with MeOH–H O (6:4, 500 ml) to give 17 (9.6 mg) and 3
(24 mg). Subfraction 23 (128 mg) was subjected to a reversed
phase column (Lichroprep RP-18, 25–40 m, 30 g) employing
MeOH–H O (6:4, 750 ml) to afford 1 (28 mg). Subfraction 24
(165 mg) was applied to a reversed phase column (Lichroprep
RP-18, 25–40 m, 30 g) using MeOH–H O (6:4, 750 ml) to give
2
lm, 30 g), employing MeOH–H O (6:4, 500 ml) yielding 15
(
l
eter. IR measurements were obtained on a Bruker IFS-48 spectrom-
eter. NMR experiments were performed on a Bruker DRX-600
spectrometer (Bruker BioSpinGmBH, Rheinstetten, Germany)
equipped with a Bruker 5 mm TCI CryoProbeat 300 K. All 2D-
2
l
2
3
NMR spectra were acquired in CD OD (99.95%, SigmaAldrich) and
standard pulse sequences and phase cycling were used for DQF-
COSY, HSQC, and HMBC spectra. The NMR data were processed
using UXNMR software. Exact masses were measured by a Voyager
DE mass spectrometer. Samples were analyzed by matrix-assisted
laser desorption ionization time-of-flight (MALDITOF) mass spec-
trometry. A mixture of analyte solution and a-cyano-4-hydroxycin-
namic acid (Sigma) was applied to the metallic sample plate and
dried. Mass calibration was performed with the ions from ACTH
l
2
13 (17.8 mg) and 11 (10 mg). Subfraction 25 (116 mg) was chro-
matographed on a reversed phase material (Lichroprep RP-18,
25–40
2
lm, 30 g), employing MeOH–H O (6:4, 600 ml) to afford 4
(16 mg) and 9 (16 mg).
3
.4. 3-O-[
b- -xylopyranosyl]-6-O-b-
hydroxycycloartane (1)
a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosyl-(1 ? 2)-
D
D-xylopyranosyl-3b,6a,16b,24(S),25-penta-
(
9
fragment 18–39) at 2465.1989 Da and angiotensin III at
31.5154 Da as internal standard. ESIMS analyses were performed
using a ThermoFinnigan LCQ Deca XP Max iontrap mass spectrom-
eter equipped with Xcalibur software. GC analysis was performed
on a Termo Finnigan Trace GC apparatus using a l-Chirasil-Val col-
umn (0.32 mm ꢀ 25 m).
25
Amorphous white solid; C51
H
86
O
21; ½
a
ꢁ
+37.2° (c 0.1 MeOH); IR
D
KBr
ꢂ1
m
cm : 3474 (>OH), 3035 (cyclopropane ring), 2953 (>CH),
max
1
3
1
750 (C@O), 1255 and 1068 (C–O–C); C NMR (CD
MHz) aglycon moiety: d 32.7 (C-1), 30.2 (C-2), 89.6 (C-3), 42.5
C-4), 52.9 (C-5), 79.0 (C-6), 34.3 (C-7), 45.6 (C-8), 21.9 (C-9),
3
OD, 150
(
3.2. Plant material
2
4
2
7
1
9.1 (C-10), 26.8 (C-11), 33.8 (C-12), 46.6 (C-13), 47.0 (C-14),
7.7 (C-15), 73.0 (C-16), 57.6 (C-17), 18.0 (C-18), 27.7 (C-19),
9.8 (C-20), 18.5 (C-21), 34.1 (C-22), 28.3 (C-23), 78.1 (C-24),
A. aureus Willd (Whole plant; Section: Adiaspastus) was col-
lected from Karlıova, Bingöl, 40 km from Çat to Karlıova, from
180 m altitude, East Anatolia, Turkey in July 2009, and identified
3.5 (C-25), 25.2 (C-26), 25.2 (C-27), 28.0 (C-28), 16.6 (C-29),
2
1
9.9 (C-30); H NMR (CD
3
OD, 600 MHz) aglycon moiety: d 4.48
1H, ddd, J = 8.0, 8.0, 5.2 Hz, H-16), 3.54 (1H, ddd, J = 9.5, 9.5,
.5 Hz, H-6), 3.41 (1H, dd, J = 10.5, 2.4 Hz, H-24), 3.23 (1H, dd,
J = 11.3, 4.0 Hz, H-3), 1.74 (1H, dd, J = 9.9, 8.0 Hz, H-17), 1.28 (3H,
s, Me-28), 1.20 (3H, s, Me-27), 1.18 (3H, s, Me-26), 1.17 (3H, s,
Me-18), 1.03 (3H, s, Me-29), 1.01 (3H, s, Me-30), 0.98 (3H, d,
by Fevzi Özgökçe (Department of Biology, Faculty of Science and
Art, Yüzüncü Yıl University, Van, Turkey). Voucher specimen has
been deposited in the Herbarium of Yüzüncü Yıl University, Van,
Turkey (VANF 13646).
(
4
3.3. Extraction and isolation
J = 6.5 Hz, Me-21), 0.59 and 0.25 (each 1H, d, J = 4.2 Hz, H
2
-19);
1
13
for the H (CD
of the sugar portion see Table 1; ESI–MS m/z 1057.65 [M + Na] ;
3
OD, 600 MHz) and C NMR (CD OD, 150 MHz) data
3
Air-dried and powdered plant material of A. aureus (Whole
+
plant, 700 g) was extracted with MeOH (2 ꢀ 3.5 l) at 60 °C. After fil-
+
3
MS/MS m/z 907.53 [M + Na-150] , MS m/z 761.46 [M + Na-150–
46] , 629.40 [M + Na-150–146–132] ; HRMALDITOFMS [M + Na]
m/z 1057.5563 (calcd for C51 21Na, 1057.5559).
tration, the solvent was removed by rotary evaporation yielding
+
+
+
1
7
2 g of extract. The MeOH extract was dissolved in H
2
O (350 ml),
86
H O
and successively partitioned with hexane (2 ꢀ 200 ml), EtOAc
(
2 ꢀ 200 ml) and n-BuOH saturated with H O (3 ꢀ 200 ml). The
2
n-BuOH extract (27 g) was subjected to vacuum liquid chromatog-
3.5. 3,6-Di-O-b-D-xylopyranosyl-3b,6a,16b,24(S),25-
raphy (VLC) using reversed- phase material (Lichroprep RP-18, 25–
pentahydroxycycloartane (2)
4
1
0
l
m, 150 g) employing
2 2
H O (1000 ml), H O–MeOH (8:2,
2
D
5
200 ml; 6:4, 2400 ml; 4:6, 3200 ml; 2:8, 1600 ml) and MeOH
Amorphous white solid; C40
H
68
O
13; ½
a
ꢁ
+29.2° (c 0.1 MeOH); IR
KBr
ꢂ1
(
(
800 ml) to give seventeen main fractions (1–17). Fraction 7
3.2 g) was submitted to silica gel (330 g) column chromatography
m
cm : 3487 (>OH), 3043 (cyclopropane ring), 2950 (>CH),
max
1
13
1269 and 1059 (C–O–C); H (CD
(CD OD, 150 MHz) data of the aglycon moiety were superimpos-
able with those reported for 1; for the H (CD
3
OD, 600 MHz) and C NMR
with the solvent system CHCl
0:20:2, 2500 ml; 70:30:3, 1500 ml) yielding 7 (93.3 mg), 18
32.3 mg), 2 (25.6 mg), 16 (127.5 mg) and 25 subfractions. Subfrac-
tion 5 (150 mg) was subjected to silica gel (30 g) column chroma-
3 2
–MeOH–H O (90:10:1, 2000 ml;
3
1
8
(
3
OD, 600 MHz) and
3
OD, 150 MHz) data of the sugar portion see Table
1
3
C NMR (CD
1; ESI–MS m/z 779.54 [M + Na] , MS/MS m/z 629.38 [M + Na-
+

7
66
D. Gülcemal et al. / Phytochemistry 72 (2011) 761–768
+
+
+
1
50] , 479.38 [M + Na-150–150] ; HRMALDITOFMS [M + Na] m/z
3.9. 3-O-[a-L-arabinopyranosyl-(1?2)-b-D-xylopyranosyl]-3b,6a,16b,
7
79.4561 (calcd for C40
H
68
O13Na, 779.4558).
24a-tetrahydroxy-20(R),25-epoxycycloartane (6)
2
5
Amorphous white solid; C40
H
66
O13; ½
a
ꢁ
+10.5° (c 0.1 MeOH); IR
D
3.6. 3,6-Di-O-b-
D
-xylopyranosyl-25-O-b-
D
-glucopyranosyl-3b,6
a
,16b,
KBr
ꢂ1
m
cm : 3488 (>OH), 3053 (cyclopropane ring), 2939 (>CH),
274 and 1050 (C–O–C); C NMR (CD OD, 150 MHz) aglycon moi-
3
24(S),25-pentahydroxycycloartane (3)
max
1
3
1
2
5
ety: d 33.2 (C-1), 30.2 (C-2), 89.3 (C-3), 43.1 (C-4), 54.3 (C-5), 69.3
(C-6), 38.7 (C-7), 48.4 (C-8), 21.7 (C-9), 30.4 (C-10), 26.8 (C-11),
34.3 (C-12), 46.4 (C-13), 47.5 (C-14), 48.2 (C-15), 74.8 (C-16),
Amorphous white solid; C46
H
78
O
18; ½
a
ꢁ
+35.6° (c 0.1 MeOH); IR
D
KBr
ꢂ1
m
cm : 3482 (>OH), 3040 (cyclopropane ring), 2945 (>CH),
max
1
3
1
259 and 1051 (C–O–C); C NMR (CD
ety: d 32.6 (C-1), 30.1 (C-2), 89.6 (C-3), 42.7 (C-4), 52.7 (C-5), 79.0
C-6), 34.0 (C-7), 45.7 (C-8), 22.0 (C-9), 29.0 (C-10), 26.6 (C-11),
3
OD, 150 MHz) aglycon moi-
6
2
2
1.2 (C-17), 21.0 (C-18), 31.8 (C-19), 80.3 (C-20), 28.0 (C-21),
6.6 (C-22), 23.7 (C-23), 69.3 (C-24), 76.5 (C-25), 28.2 (C-26, C-
(
1
7, C-28), 16.2 (C-29), 20.4 (C-30); H NMR (CD
3
OD, 600 MHz)
3
5
3
2
6
1
2
3.7 (C-12), 46.3 (C-13), 47.2 (C-14), 47.4 (C-15), 72.9 (C-16),
7.6 (C-17), 17.9 (C-18), 27.4 (C-19), 30.0 (C-20), 18.3 (C-21),
4.0 (C-22), 28.4 (C-23), 76.5 (C-24), 81.2 (C-25), 22.4 (C-26),
aglycon moiety: d 4.64 (1H, ddd, J = 8.0, 8.0, 5.2 Hz, H-16), 3.47
1H, ddd, J = 9.5, 9.5, 4.5 Hz, H-6), 3.51 (1H, br s, H-24), 3.22 (1H,
dd, J = 11.3, 4.0 Hz, H-3), 2.01 (1H, dd, J = 9.9, 8.0 Hz, H-17), 1.54
3H, s, Me-21), 1.49 (3H, s, Me-18), 1.32 (6H, s, Me-28, Me-27),
.22 (3H, s, Me-26), 1.05 (3H, s, Me-29), 0.97 (3H, s, Me-30), 0.57
(
1
3.0 (C-27), 28.0 (C-28), 16.3 (C-29), 19.6 (C-30); H NMR (CD
3
OD,
(
00 MHz) aglycon moiety: d 4.48 (1H, ddd, J = 8.0, 8.0, 5.2 Hz, H-
6), 3.54 (1H, ddd, J = 9.5, 9.5, 4.5 Hz, H-6), 3.61 (1H, dd, J = 10.5,
.4 Hz, H-24), 3.23 (1H, dd, J = 11.3, 4.0 Hz, H-3), 1.73 (1H, dd,
J = 9.9, 8.0 Hz, H-17), 1.27 (3H, s, Me-28), 1.29 (3H, s, Me-27),
.25 (3H, s, Me-26), 1.17 (3H, s, Me-18), 1.03 (3H, s, Me-29), 1.01
3H, s, Me-30), 0.97 (3H, d, J = 6.5 Hz, Me-21), 0.59 and 0.24 (each
1
1
and 0.43 (each 1H, d, J = 4.2 Hz, H
2
-19); for the H (CD
3
OD,
1
3
6
00 MHz) and C NMR (CD
tion see Table 2; ESI–MS m/z 777.74 [M + Na] , MS/MS m/z
3
OD, 150 MHz) data of the sugar por-
+
1
+
+
6
45.36 [M + Na-132] , 495.41 [M + Na-132–150] ; HRMALDI-
(
+
1
13
66
TOFMS [M + Na] m/z 777.4405 (calcd for C40H O13Na, 777.4401).
1
H, d, J = 4.2 Hz, H
2 3
-19); for the H (CD OD, 600 MHz) and C NMR
(
CD
3
OD, 150 MHz) data of the sugar portion see Table 1; ESI–MS m/
+
+
3
z 941.59 [M + Na] , MS/MS m/z 791.43 [M + Na-150] , MS m/z
3.10. 6-O-b-D-glucopyranosyl-3b,6a,16b,24a-tetrahydroxy-20(R),25-
+
4
6
1
C
11.37 [M + Na-150–180] , MS m/z 479.31 [M + Na-150–180–
epoxycycloartane (7)
+
+
32] ; HRMALDITOFMS [M + Na] m/z 941.5090 (calcd for
18Na, 941.5086).
H
78
O
25
D
46
Amorphous white solid; C36
60
H O
10; ½
aꢁ
+15.2° (c 0.1 MeOH); IR
KBr
ꢂ1
m
cm : 3479 (>OH), 3048 (cyclopropane ring), 2947 (>CH),
max
1
3
3.7. 3-O-b-
D
-xylopyranosyl-6,25-di-O-b-
D
-glucopyranosyl-3b,6
a
,16b,
1257 and 1065 (C–O–C); C NMR (CD
ety: d 33.2 (C-1), 31.0 (C-2), 79.1 (C-3), 42.2 (C-4), 52.7 (C-5), 80.2
C-6), 35.1 (C-7), 46.8 (C-8), 21.8 (C-9), 29.9 (C-10), 26.3 (C-11),
3
OD, 150 MHz) aglycon moi-
24(S),25-pentahydroxycycloartane (4)
(
2
D
5
Amorphous white solid; C47
H
80
O
19; ½
a
ꢁ
+32.5° (c 0.1 MeOH); IR
34.3 (C-12), 46.5 (C-13), 47.7 (C-14), 47.7 (C-15), 74.9 (C-16),
60.8 (C-17), 21.0 (C-18), 30.1 (C-19), 80.2 (C-20), 28.3 (C-21),
KBr
ꢂ1
m
cm : 3477 (>OH), 3038 (cyclopropane ring), 2934 (>CH),
max
1
13
1
263 and 1054 (C–O–C); H (CD
CD OD, 150 MHz) data of the aglycon moiety were superimpos-
able with those reported for 3; for the H (CD
3
OD, 600 MHz) and C NMR
26.5 (C-22), 23.7 (C-23), 69.4 (C-24), 76.1 (C-25), 28.2 (C-26),
1
(
3
28.3 (C-27, C-28), 15.4 (C-29), 19.9 (C-30); H NMR (CD
3
OD,
1
OD, 600 MHz) and
3
OD, 150 MHz) data of the sugar portion see Table
600 MHz) aglycon moiety: d 4.64 (1H, ddd, J = 8.0, 8.0, 5.2 Hz, H-
16), 3.58 (1H, ddd, J = 9.5, 9.5, 4.5 Hz, H-6), 3.51 (1H, br s, H-24),
3.24 (1H, dd, J = 11.3, 4.0 Hz, H-3), 2.02 (1H, dd, J = 9.9, 8.0 Hz, H-
17), 1.54 (3H, s, Me-21), 1.47 (3H, s, Me-18), 1.32 (3H, s, Me-27),
1.27 (3H, s, Me-28), 1.22 (3H, s, Me-26), 0.98 (3H, s, Me-29), 0.99
3
1
3
C NMR (CD
; ESI–MS m/z 971.56 [M + Na] , MS/MS m/z 791.46 [M + Na-
+
1
1
+
3
+
4
80] , MS m/z 611.40 [M + Na-180–180] , MS m/z 479.32
+
+
[
M + Na-180–180–132] ;
71.5197 (calcd for C47
HRMALDITOFMS
19Na, 971.5192).
[M + Na]
m/z
9
H
80
O
(3H, s, Me-30), 0.62 and 0.35 (each 1H, d, J = 4.2 Hz, H
2
-19); for
1
13
the H (CD
of the sugar portion see Table 1; ESI–MS m/z 675.47 [M + Na] ,
3
OD, 600 MHz) and C NMR (CD OD, 150 MHz) data
3
+
3
.8. 6-O-b-
oartane (5)
D-glucopyranosyl-3b,6a,16b,24(S),25-pentahydroxycycl-
+
+
MS/MS m/z 495.29 [M + Na-180] ; HRMALDITOFMS [M + Na] m/z
75.4089 (calcd for C36 10Na, 675.4084).
6
60
H O
2
5
Amorphous white solid; C36
H
62
O
10; ½
aꢁ
+ 27.8° (c 0.1 MeOH);
D
KBr
ꢂ1
IR
1
m
cm : 3485 (>OH), 3047 (cyclopropane ring), 2930 (>CH),
max
1
3
254 and 1061 (C–O–C); C NMR (CD
ety: d 32.7 (C-1), 30.7 (C-2), 79.3 (C-3), 42.6 (C-4), 52.9 (C-5), 80.1
C-6), 34.8 (C-7), 46.6 (C-8), 22.3 (C-9), 29.7 (C-10), 26.6 (C-11),
3
OD, 150 MHz) aglycon moi-
Table 2
1
H NMR data (J in Hz) of the sugar portions of compounds 6 and 8 (600 MHz, d ppm, in
CD OD).
3
(
3
5
3
2
6
3.8 (C-12), 46.6 (C-13), 47.9 (C-14), 47.9 (C-15), 73.0 (C-16),
7.7 (C-17), 18.5 (C-18), 29.0 (C-19), 29.6 (C-20), 18.3 (C-21),
6
8
d
C
d
H
(J in Hz)
d
C
H
d (J in Hz)
3.7 (C-22), 28.2 (C-23), 78.2 (C-24), 73.8 (C-25), 25.2 (C-26),
b-
D
-Xyl (at C-3)
b-D-Xyl (at C-6)
1
5.5 (C-27), 28.4 (C-28), 15.7 (C-29), 19.9 (C-30); H NMR (CD
3
OD,
1
2
3
4
5
105.2
82.9
76.3
70.5
65.7
4.48, d (7.5)
3.46, dd (9.2, 7.5)
3.55, t (9.2)
3.54, m
3.88, dd (11.7, 5.2)
104.9
74.9
77.7
70.9
66.2
4.33, d (7.5)
3.20, dd (9.2, 7.5)
3.32, t (9.2)
3.49, m
3.86, dd (11.7, 5.2)
3.20, t (11.7)
00 MHz) aglycon moiety: d 4.47 (1H, ddd, J = 8.0, 8.0, 5.2 Hz,
H-16), 3.58 (1H, ddd, J = 9.5, 9.5, 4.5 Hz, H-6), 3.41 (1H, dd,
J = 10.5, 2.4 Hz, H-24), 3.25 (1H, dd, J = 11.3, 4.0 Hz, H-3), 1.74
(
1H, dd, J = 9.9, 8.0 Hz, H-17), 1.25 (3H, s, Me-28), 1.19 (6H, s,
3
.22, t (11.7)
Me-26, Me-27), 1.18 (3H, s, Me-18), 0.98 (3H, s, Me-29), 1.01
a
-
L
-Ara (at C-2xyl
)
(
3H, s, Me-30), 0.98 (3H, d, J = 6.5 Hz, Me-21), 0.61 and 0.30 (each
1
2
3
4
5
105.9
73.0
73.7
69.1
66.8
4.49, d (3.7)
3.68, dd (8.5, 3.7)
3.59, dd (8.5, 3.0)
3.89, m
3.91, dd (11.9, 2.0)
3
1
13
1
H, d, J = 4.2 Hz, H
2 3
-19); for the H (CD OD, 600 MHz) and C NMR
(
CD
m/z 677.56 [M + Na] , MS/MS m/z 497.37 [M + Na-180] ; HRMAL-
DITOFMS [M + Na]+ m/z 677.4246 (calcd for
10Na,
77.4241).
3
OD, 150 MHz) data of the sugar portion see Table 1; ESI–MS
+
+
C H
36 62
O
.55, dd (11.9, 3.0)
6

D. Gülcemal et al. / Phytochemistry 72 (2011) 761–768
767
3.11. 6-O-b-
D-xylopyranosyl-3b,6
a,16b,24a-tetrahydroxy-20(R),25-
References
epoxycycloartane (8)
Agzamova, M.A., Isaev, M.I., 1999. Triterpene glycosides of Astragalus and their
genins LIX. Structure of cyclocanthoside F. Chem. Nat. Compd. 35, 314–319.
Agzamova, M.A., Isaev, M.I., 1998. Triterpene glycosides of Astragalus and their
genins. LVI. Cyclopycnanthoside – A new cycloartane glycoside. Chem. Nat.
Compd. 34, 155–159.
2
5
Amorphous white solid; C35
H
58
O
9
; ½
a
ꢁ
+8.2° (c 0.1 MeOH); IR
D
KBr
max
ꢂ1
m
cm : 3471 (>OH), 3050 (cyclopropane ring), 2943 (>CH),
1
13
1
268 and 1057 (C–O–C); H (CD
3
OD, 600 MHz) and C NMR
_
Bedir, E., Çalı ßs , I., Aquino, R., Piacente, S., Pizza, C., 1998a. Cycloartane triterpene
(
3
CD OD, 150 MHz) data of the aglycon moiety were superimpos-
glycosides from the roots of Astragalus brachypterus and Astragalus
1
able with those reported for 7; for the H (CD
3
OD, 600 MHz) and
OD, 150 MHz) data of the sugar portion see Table
microcephalus. J. Nat. Prod. 61, 1469–1472.
1
3
_
C NMR (CD
; ESI–MS m/z 645.50 [M + Na] , MS/MS m/z 495.30 [M + Na-
3
Bedir, E., Çalı sß , I., Zerbe, O., Sticher, O., 1998b. Cyclocephaloside I:
a
novel
+
cycloartane-type glycoside from Astragalus microcephalus. J. Nat. Prod. 61,
03–505.
2
1
C
5
+
+
50] ; HRMALDITOFMS [M + Na] m/z 645.3983 (calcd for
Na, 645.3979).
_
Bedir, E., Çalı sß , I., Aquino, R., Piacente, S., Pizza, C., 1999. Trojanoside H:
a
58 9
H O
cycloartane-type secondary glycoside from the aerial parts of Astragalus
trojanus. Phytochemistry 51, 1017–1020.
35
_
Bedir, E., Pugh, N., Çalı sß , I., Pasco, D.S., Khan, I.A., 2000a. Immunostimulatory effects
3.12. Acid hydrolysis
of cycloartane-type triterpene glycosides from Astragalus species. Biol. Pharm.
Bull. 23, 834–837.
_
Bedir, E., Çalı ßs , I., Khan, I.A., 2000b. Macrophyllosaponin E: a novel compound from
The configuration of sugar units were established after hydroly-
the roots of Astragalus oleifolius. Chem. Pharm. Bull. 48, 1081–1083.
Breitmaier, E., Voelter, W., 1987. Carbon-13 NMR Spectroscopy. VCH
Verlagsgellsschaft Geulsh, Weinheim, Germany. pp. 380–393.
sis of 1 and 3 with 1 N HCl, trimethylsilation, and determination of
the retention times by GC operating in the experimental conditions
previously reported by De Marino et al., 2003.
_
_
Çalı sß , I., Zor, M., Saraco g˘ lu, I., I sß imer, A., Rüegger, H., 1996. Four novel cycloartane
glycosides from Astragalus oleifolius. J. Nat. Prod. 59, 1019–1023.
The peaks of the hydrolysate of 1 were detected at 8.94 and
Çalı sß , I_ ., Yürüker, A., Ta sß demir, D., Wright, A.D., Sticher, O., Luo, Y.D., Pezzuto, J.M.,
9
.81 min (
and 12.02 (
and -glucose (14.73 min) were detected in the hydrolysate of 3.
L
-arabinose), 9.70 and 10.72 (
-xylose). The peaks of -xylose (10.98 and 12.01 min)
L
-rhamnose) and 10.99
1997. Cycloartane triterpene glycosides from the roots of Astragalus
melanophrurius. Planta Med. 63, 183–186.
D
D
Davis, P.H., 1970. Flora of Turkey and East Aegean Island, vol. 4. University Press,
Edinburgh. pp. 49–254.
D
Retention times for authentic samples after being treated in the
same manner with 1-(trimethylsilyl)-imidazole in pyridine were
De Marino, S., Borbone, N., Iorizzi, M., Esposito, G., McClintock, J.B., Zollo, F., 2003.
Bioactive asterosaponins from the starfish Luidia quinaria and Psilaster cassiope.
Isolation and structure characterization by two-dimensional NMR
spectroscopy. J. Nat. Prod. 66, 515–519.
detected at 8.92 and 9.80 (
L-arabinose), 9.67 and 10.70 (
L-rham-
nose), 10.98 and 12.00 min (
D
-xylose), and 14.71 min ( -glucose).
D
Fadeev, Y.M., Isaev, M.I., Akimov, Y.A., Kintya, P.K., Gorovits, M.B., Abubakirov, N.K.,
1988. Triterpene glycosides and their genins from Astragalus. XXV.
Cyclocanthoside D from Astragalus tragacantha. Khim. Prir. Soedin. 1, 73–
76.
3.13. Antiproliferative activity
Gariboldi, P., Pelizzoni, F., Tatò, M., Verotta, L., El-Sebakhy, N.A., Asaad, A.M.,
Abdallah, R.M., Toaima, S.M., 1995. Cycloartane triterpene gylcosides from
Astragalus trigonus. Phytochemistry 40, 1755–1760.
Hirotani, M., Zhou, Y., Rui, H., Furuya, T., 1994a. Cycloartane triterpene glycosides
from the hairy root cultures of Astragalus membranaceus. Phytochemistry 37,
HL-60 (human promyelocytic leukemia), MCF-7 (human breast
cancer), HT-29 (human colon carcinoma), A549 (human lung ade-
nocarcinoma), PC3 (human prostate cancer) cells obtained from
the European Collection of Cell Cultures were cultured in DMEM
1403–1407.
Hirotani, M., Zhou, Y., Lui, H., Furuya, T., 1994b. Astragaloside from hairy root
medium supplemented with 2 mM
vated fetal bovine serum (FBS), 1% penicillin/streptomycin at
7 °C in an atmosphere of 95% O and 5% CO . The cells were used
up to a maximum of 10 passages.
L-glutamine, 10% heat-inacti-
cultures of Astragalus membranaceus. Phytochemistry 36, 665–670.
_
Horo, I., Bedir, E., Perrone, A., Özgökçe, F., Piacente, S., Alanku sß -Çalı sß kan, Ö., 2010.
Triterpene glycosides from Astragalus icmadophilus. Phytochemistry 71, 956–
3
2
2
963.
Isaev, M.I., Imomnazarov, B.A., Fadeev, Y.M., Kintya, P.A., 1992. Triterpene
glycosides of Astragalus and their genins. XLII. Cycloartanes of Astragalus
tragacantha. Khim. Prir. Soedin. 3 (4), 360–367.
Isaev, M.I., Gorovits, M.B., Gorovits, T.T., Abdullaev, N.D., Abubakirov, N.K., 1983.
Astragalus triterpenoid glycosides and their genins. VIII. Askendoside C from
Astragalus taschkendicus. Khim. Prir. Soedin. 2, 173–180.
3
Human cancer cells (3 ꢀ 10 ) were plated in 96-well culture
plates in 90 L of culture medium and incubated at 37 °C in
humidified 5% CO . The next day, 10 L aliquots of serial dilutions
of each test compound (1–50 M) and etoposide, used as positive
control, were added to the cells and incubated for 48 h. Cell viabil-
ity was assessed through the MTT assay. Briefly, 25 L of MTT
5 mg/mL) were added and the cells were incubated for an addi-
tional 3 h. Thereafter, cells were lysed and the dark blue crystals
solubilized with 100 L of a solution containing 50% N,N-dimethyl-
l
2
l
l
Isaev, M.I., Gorovits, M.B., Abdullaev, N.D., Abubakirov, N.K., 1982. Triterpenoid
glycosides of Astragalus and their genins. VI. Cycloasgenin C from Astragalus
taschkendicus. Khim. Prir. Soedin. 4, 458–464.
l
Kasai, E., Ohikara, M., Asakawa, J., Mizutani, K., Tanaka, O., 1979. 13C NMR Study of
(
a
- and b-anomeric pairs of
Dmannopyranosides and L-rhamnopyranosides.
Tetrahedron 35, 1427–1432.
l
Kikuchi, T., Akihisa, T., Tokuda, H., Ukiya, M., Watanabe, K., Nishino, H., 2007. Cancer
chemopreventive effects of cycloartane-type and related triterpenoids in vitro
and in vivo models. J. Nat. Prod. 70, 918–922.
formamide, 20% SDS (Sodium Dodecyl Sulfate) with an adjusted pH
of 4.5. The optical density (OD) of each well was measured with a
microplate spectrophotometer (Titertek Multiskan MCC/340)
equipped with a 620 nm filter. Cells viability in response to treat-
ment was calculated as percentage of control cells treated with sol-
vent DMSO at the final concentration 0.1%:% viable cells = (100 ꢀ
OD treated cells)/OD control cells.
Nian, Y., Zhang, Y.-L., Chen, J.-C., Lu, L., Qiu, M.-H., Qing, C., 2010. Cytotoxic chemical
constituents from the roots of Cimicifuga fetida. J. Nat. Prod. 73, 93–98.
_
Özipek, M., Dönmez, A.A., Çalı ßs , I., Brun, R., Rüedi, P., Tasdemir, D., 2005.
Leishmanicidal cycloartane-type triterpene glycosides from Astragalus
oleifolius. Phytochemistry 66, 1168–1173.
Polat, E., Alanku sß -Çalı ßs kan, Ö., Perrone, A., Piacente, S., Bedir, E., 2009. Cycloartane-
type glycosides from Astragalus amblolepis. Phytochemistry 70, 628–634.
Polat, E., Bedir, E., Perrone, A., Piacente, S., Alanku sß -Çalı ßs kan, Ö., 2010. Triterpenoid
saponins from Astragalus wiedemannianus. Phytochemistry 71, 658–662.
Ríos, L.J., Waterman, P.G., 1997. A review of the pharmacology and toxicology of
Astragalus. Phytother. Res. 11, 411–418.
Acknowledgements
Semmar, N., Fenet, B., Lacaille-Dubois, M.-A., Gluchoff-Fiasson, K., Chemli, R., Jay,
M., 2001. Two new glycosides from Astragalus caprinus. J. Nat. Prod. 64, 656–
This project was supported by TUBITAK (109T425), Ege Univer-
sity Research Foundation (2009 Fen 045) and EBILTEM (2010/BIL/
658.
0
08).
Sukhina, I.A., Mamedova, R.P., Agzamova, M.A., Isaev, M.I., 2007. Triterpene
glucosides of Astragalus and their genins. LXXIV. Cyclotrisectoside, the first
trisdesmoside of cyclocephalogenin. Chem. Nat. Compd. 43, 159–161.
Tang, W., Eisenbrand, G., 1992. In Chinese Drugs of Plant Origin. Springer-Verlag,
Berlin. pp. 191–197.
Appendix A. Supplementary data
Tian, Z., Yang, M., Huang, F., Li, K., Si, J., Shi, L., Chen, S., Xiao, P., 2005. Cytotoxicity of
three cycloartane triterpenoids from Cimicifuga dahurica. Cancer Lett. 226, 65–
75.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2011.02.006.

7
68
D. Gülcemal et al. / Phytochemistry 72 (2011) 761–768
_
Verotta, L., El-Sebakhy, N.A., 2001. Studies in Natural Products Chemistry: Bioactive
Natural Products, vol. 25. Elsevier, Amsterdam. Part F, pp. 179–234.
Watanabe, K., Mimaki, Y., Sakagami, H., Sashida, Y., 2002. Cycloartane glycosides
from the rhizomes of Cimicifuga racemosa and their cytotoxic activities. Chem.
Pharm. Bull. 50, 121–125.
Ye sß ilada, E., Bedir, E., Çalı ßs , I., Takaishi, Y., Ohmoto, Y., 2005. Effects of triterpene
saponins from Astragalus species on in vitro cytokine release. J.
Ethnopharmacol. 96, 71–77.