Cycloartane glycosides from Astragalus campylosema Boiss. ssp. campylosema

Article abstract of DOI:10.1016/j.phytochem.2008.08.002

Four cycloartane glycosides, 3-O-[α-l-arabinopyranosyl-(1 → 2)-β-d-xylopyranosyl]-3β,6α,16β,23α,25-pentah ydroxy-20(R),24(S)-epoxycycloartane (1), 3-O-[α-l-arabinopyranosyl-(1 → 2)-β-d-xylopyranosyl]-16-O-hydroxyacetoxy-23-O-acetoxy-3β, 6α,25-trihydroxy-2

Full text of DOI:10.1016/j.phytochem.2008.08.002

Phytochemistry 69 (2008) 2634–2638
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Cycloartane glycosides from Astragalus campylosema Boiss. ssp. campylosema
a,d,
b
c
c
c,
_
*
*
Ihsan Çalısß
, Ali A. Dönmez , Angela Perrone , Cosimo Pizza , Sonia Piacente
a Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, TR-06100 Ankara, Turkey
b Department of Biology, Faculty of Science, Hacettepe University, TR-06532 Ankara, Turkey
c Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, I-84084 Fisciano, Italy
d Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmacy, Near East University, Nicosia, Turkish Republic of Northern Cyprus
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 April 2008
Received in revised form 4 August 2008
Available online 18 September 2008
Four cycloartane glycosides, 3-O-[
25-pentahydroxy-20(R),24(S)-epoxycycloartane
a
-
L
-arabinopyranosyl-(1 ? 2)-b-
(1), 3-O-[ -arabinopyranosyl-(1 ? 2)-b-
pyranosyl]-16-O-hydroxyacetoxy-23-O-acetoxy-3b,6 ,25-trihydroxy-20(R),24(S)-epoxycycloartane (2),
3-O[ -arabinopyranosyl-(1 ? 2)-b- -xylopyranosyl]-3b,6 ,23 ,25-tetrahydroxy-20(R),24(R)-16b,24;20,
24-diepoxycycloartane (3), 3-O-[ -arabinopyranosyl-(1 ? 2)-b- -xylopyranosyl]-25-O-b- -glucopyr-
anosyl-3b,6 ,16b,25-tetrahydroxy-20(R),24(S)-epoxycycloartane (4), along with three known cycloar-
D
-xylopyranosyl]-3b,6
a
,16b,23
a,
a
-
L
D-xylo-
a
a
-L
D
a
a
a
-
L
D
D
Keywords:
a
Astragalus campylosema ssp. campylosema
Leguminosae
20,24-Epoxycycloartane glycosides
16b,24;20,24-Diepoxycycloartane
tane glycosides were isolated from the MeOH extract of the roots of Astragalus campylosema ssp.
campylosema. Their structures were established by the extensive use of 1D- and 2D-NMR experiments
along with ESIMS and HRMS analysis. The occurrence of the hydroxyl function at position 23 (1–2)
and of the ketalic function at C-24 (3) are very unusual findings in the cycloartane class.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
tic and tonic and for the treatment of nephritis, diabetes, leukemia,
uterine cancer (Çalıßs et al., 1996).
The genus Astragalus L. is one of the largest and most widely dis-
tributed genera belonging to the family Leguminosae, comprising
380 species distributed mainly in the flora of Turkey (Davis,
1970). Astragalus species, growing wild in Turkey, are economically
important for the production of gum tragacanth, a very well-
known foodstuff and pharmaceutical emulsifier (Çalıßs and Sticher,
1996). In Turkish folk medicine, the aqueous extracts of some
Astragalus species (undeclared by the healer) are used to treat leu-
kaemia as well as for wound healing (Çalıßs et al., 1997; Bedir et al.,
2000). Roots of these plants are also used as antiperspirant, diure-
Polysaccharides and saponins are the major classes of chem-
ical compounds isolated from Astragalus species. The former
compounds are reported to possess cytotoxic and immunostim-
ulating effects (Ríos and Waterman, 1997; Bedir et al., 2000;
Li, 2000) but the saponins are the class of constituents more
investigated. In the course of studies on Turkish Astragalus spe-
cies several cycloartane- and oleanane-type triterpene glycosides
were isolated and their structures were elucidated (Bedir et al.,
1998a,b, 1999a,b, 2000; Çalıßs et al., 1999, 1997, 1996; Özipek
et al., 2005). Cycloartane- and oleanane-type glycosides from
Astragalus species show interesting biological properties, includ-
ing immunostimulating (Yesilada et al., 2005; Çalıßs et al., 1997;
Bedir et al., 2000), anti-protozoal (Özipek et al., 2005), antiviral
(Gariboldi et al., 1995) and cytotoxic activities (Radwan et al.,
2004).
As a part of our ongoing research of new bioactive com-
pounds from Turkish Astragalus species, we have investigated
the roots of Astragalus campylosema Boiss. (Leguminosae). This
paper reports the isolation of four new cycloartane glycosides
(1–4) along with three known compounds (5–7). Their struc-
tures were elucidated by extensive spectroscopic methods
including 1D- (1H, 13C and TOCSY) and 2D-NMR (DQF-COSY,
HSQC, HMBC, and ROESY) experiments as well as ESIMS
analysis.
* Corresponding authors. Tel.: +90 (312) 3051089; fax: +90 (312) 3114777
_
(I. Çalıßs), tel.: +39 089 969763; fax: +39 089 969602 (S. Piacente).
_
E-mail addresses: icalis@hacettepe.edu.tr (I. Çalısß), piacente@unisa.it (S. Pia-
cente).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.08.002

_
I. Çalıßs et al. / Phytochemistry 69 (2008) 2634–2638
2635
Table 1
R2
13C and 1H NMR data (J in Hz) of the sugar portions of compounds 1–4 (600 MHz,
CD3OD)
O
OR1
1–3
b- -Xyl
4.48 d (7.5)
4
OR3
D
b-
D-Xyl
1
2
3
4
5
105.8
83.2
76.7
70.8
65.8
105.6
83.1
76.7
70.7
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)
3.22 t (11.7)
β-D-xyl
3.46 dd (9.2, 7.5)
3.55 t (9.2)
3.54 m
3.89 dd (11.7, 5.2)
3.22 t (11.7)
O
O
HO
HO
OH
OH
O
O
a
-
L-Ara
a-
L
-Ara
HO
1
2
3
4
5
106.4
73.4
73.9
69.3
67.0
4.50 d (6.8)
106.2
73.3
73.8
69.3
66.9
4.50 d (6.8)
OH
3.68 dd (8.5, 6.8)
3.59 dd (8.5, 3.0)
3.83 m
3.92 dd (11.9, 2.0)
3.55 dd (11.9, 3.0)
3.68 dd (8.5, 6.8)
3.59 dd (8.5, 3.0)
3.83 m
3.92 dd (11.9, 2.0)
3.56 dd (11.9, 3.0)
-Glc
4.55 d (7.5)
3.17 dd (9.0, 7.5)
3.36 t (9.0)
3.35 t (9.0)
α-L-ara
1
2
4
R1 = R3 = H
R2 = OH
b-D
1
2
3
4
5
6
98.2
74.8
77.9
71.1
77.3
62.4
R1 = COCH2OH
R1 = R2 = H
R2 =OCOCH3 R3 = H
R3 = β-D-glc
3.27 ddd (9.0, 4.5, 2.5)
3.84 dd (11.0, 2.5)
3.68 dd (11.0, 4.5)
OH
O
1.28, 1.27, 1.05 and 1.02, and five methine proton signals at d
4.68 (ddd, J = 8.0, 8.0, 5.2 Hz), 4.47 (ddd, J = 9.3, 6.3, 3.4 Hz), 3.59
(d, J = 6.3 Hz), 3.48 (ddd, J = 9.5, 9.5, 4.5 Hz) and 3.24 (dd, J = 11.3,
4.0 Hz) which were indicative of secondary alcoholic functions (Ta-
ble 3). On the basis of DQF-COSY, HSQC and HMBC spectra and by
comparison of these data with those of cyclostragenol (Kitagawa
et al., 1983), it was observed that the aglycon of compound 1 dif-
fers from cycloastragenol only by the presence of an additional sec-
ondary alcoholic function at C-23 (dH 4.47, dC 74.2). The
O
OH
β-D-xyl
O
O
HO
HO
OH
OH
O
O
HO
OH
3
α-L-ara
a-orientation of hydroxyl group on C-23 was derived by the ROESY
spectrum, which showed key correlation peaks between H-23 (d
4.47) and H-22b (d 3.09) signals and between Me-21 (d 1.42) and
2. Results and discussion
H-22
new aglycon of 1 was identified as 3b,6
oxy-20(R),24(S)-epoxycycloartane and compound 1 as the new
3-O-[ -arabinopyranosyl-(1 ? 2)-b- -xylopyranosyl]-3b,6 ,16b,
23 ,25-pentahydroxy-20(R),24(S)-epoxycycloartane.
The molecular formula of compound 2 was established as
a
(d 4.47), H-24 (d 4.47) and H-17 (d 2.34) signals. Thus, the
a
,16b,23 ,25-pentahydr-
a
Compounds 1–3. A detailed comparison of the sugar region
NMR data (1H, 13C, 1D-TOCSY, HSQC, HMBC, DQF-COSY) and ESIMS
data of compounds 1–3 showed that the disaccharide chain was
identical in the three compounds. In particular for the sugar por-
tion, compound 1 showed in the 1H NMR spectrum signals corre-
sponding to two anomeric protons at d 4.48 (1H, d, J = 7.5 Hz)
and 4.50 (1H, d, J = 6.8 Hz) (Table 1). Complete assignments of
the 1H and 13C NMR signals of the sugar portion were accom-
plished by 1D-TOCSY, HSQC, HMBC and DQF-COSY experiments
which led to the identification of one b-xylopyranosyl unit (d
a
-
L
D
a
a
C44H70O17 by HRMALDITOF mass spectrum (m/z 893.4511
[M+Na]+, calcd for C44H70O17Na, 893.4519). The positive ESIMS
spectrum showed the sodiated ion peak at m/z 893 [M+Na]+. Its
MS/MS fragmentation showed peaks at m/z 817 [M+Naꢀ76]+, due
to the loss of an hydroxyacetate molecule. The MS3 fragmentation
showed peaks at m/z 757 [M+Naꢀ76ꢀ60]+, corresponding to the
loss of an acetate molecule, at m/z 685 [M+Naꢀ76ꢀ132]+, ascrib-
able to the loss of an arabinopyranosyl unit and at m/z 305
[M+Naꢀ76ꢀ512]+, due to the loss of the aglycon moiety.
4.48) and one a-arabinopyranosyl unit (d 4.50). The determination
of the sequence and linkage sites was obtained from the HMBC
correlations between the proton signals at d 4.48 (H-1xyl) and the
carbon resonance at d 89.5 (C-3), and the proton signal at d 4.50
(H-1ara) and the carbon resonance at d 83.2 (C-2xyl). Thus, the sugar
The 1H NMR spectrum for the aglycon portion of 2 in compari-
son to that 1 showed in addition the presence of a signal at d 2.08
and two signals at d 4.24 (1H, d, J = 16.8 Hz) and 4.11 (1H, d,
J = 16.8 Hz). Moreover a detailed analysis of the 13C NMR data
and the HSQC and HMBC correlations suggested that aglycon of 2
differed from 1 only by the presence of a –COCH3 and a –COCH2OH
group (Tables 2 and 3). The HMBC correlations between the proton
signal at d 5.48 (H-16) and the carbon resonance at d 173.8 (CO-
CH2OH) and the proton signal at d 5.18 (H-23) and the carbon res-
sequence of compounds 1–3 was established as 3-O-
pyranosyl-(1 ? 2)-b- -xylopyranoside.
a-L-arabino-
D
The HRMALDITOF mass spectrum of 1 (m/z 793.4355 [M+Na]+,
calcd for C40H66O14Na, 793.4350) supported a molecular formula
of C40H66O14. The ESIMS mass spectrum showed the sodiated ion
peak at m/z 793 which was assigned to [M+Na]+. The MS/MS of this
ion showed peaks at m/z 661 [M+Naꢀ132]+, corresponding to the
loss of an arabinopyranosyl unit, at m/z 511 [M+Naꢀ132ꢀ150]+,
ascribable to the loss of a xylopyranosyl unit and at m/z 305
[M+Naꢀ488]+, due to the loss of the aglycon moiety.
onance at
d 172.1 (COCH3) confirmed the location of the
–COCH2OH group at C-16 and the –COCH3 group at C-23, respec-
tively. Thus, the structure of new compound 2 was established as
The 1H NMR spectrum showed for the aglycon moiety signals
due to a cyclopropane methylene at d 0.58 and 0.42 (each 1H, d,
J = 4.2 Hz), seven tertiary methyl groups at d 1.42, 1.33, 1.30,
3-O-[
a
-
L
-arabinopyranosyl-(1 ? 2)-b-
D-xylopyranosyl]-16-O-hydro-
xyacetoxy-23-O-acetoxy-3b,6a,16b,23a,25-pentahydroxy-20(R),24(S)-
epoxycycloartane.

_
2636
I. Çalıßs et al. / Phytochemistry 69 (2008) 2634–2638
Table 2
Astragalus alopecurus (Agzamova and Isaev, 1995), Souliea vaginata
and Beesia calthaefolia (Sakurai et al., 1990).
13C NMR data of the aglycon moieties of compounds 1–4 (600 MHz, CD3OD)
Compound 4 showed in the positive ESIMS a sodiated ion peak at
m/z 939 [M+Na]+ and a significant fragment in MS/MS analysis at m/
z 759 [M+Naꢀ180]+, ascribable to the loss of an hexose unit. The
MS3 fragmentation showed peaks at m/z 627 [M+Naꢀ180ꢀ132]+
and m/z 477 [M+Naꢀ180ꢀ132ꢀ150]+, corresponding to the step-
wise loss of two pentose units, and at m/z 305 [M+Naꢀ180ꢀ454]+,
due to the loss of the aglycon moiety. Its molecular formula was
established unequivocally as C46H76O18 by HRMALDITOF mass
spectrum (m/z 939.4934 [M+Na]+, calcd for C46H76O18Na,
939.4929). The NMR data of the sugar region of 4 in comparison
with those of compounds 1–3 showed the presence of an additional
anomeric signal at d 4.55 (d, J = 7.5 Hz). On the basis of 1D-TOCSY,
HSQC, HMBC, DQF-COSY correlations this sugar unit was identified
as b-glucopyranose (Table 1). The 1H and 13C NMR data of 4 indi-
cated cycloastragenol (Kitagawa et al., 1983) as aglycon with gly-
cosidation shifts for C-3 (d 89.4) and C-25 (d 79.8) (Table 2 and 3).
Key correlation peaks in the HMBC spectrum were observed be-
tween H-1xyl (d 4.48) and C-3 (d 89.4), H-1ara (d 4.50) and C-2xyl (d
83.1) and between H-1glc (d 4.55) and C-25 (d 79.8). Therefore, the
Position
1
2
3
4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
33.1
30.5
89.5
42.9
54.6
69.2
38.8
48.5
21.5
30.2
26.6
33.7
45.3
46.4
46.5
74.3
59.4
21.9
31.9
87.9
29.7
46.6
74.2
90.4
71.5
26.1
27.7
28.4
15.9
20.0
–
33.2
30.3
89.4
43.0
54.3
69.1
38.5
48.3
21.2
30.1
26.7
33.3
46.3
46.7
46.8
77.6
58.5
21.1
31.7
86.0
28.3
44.8
76.0
85.6
71.0
26.0
27.0
28.3
16.0
20.5
33.1
30.4
89.3
43.0
54.6
69.3
39.1
48.0
21.3
30.1
27.2
34.2
45.0
45.8
43.7
75.5
61.7
23.5
33.1
84.6
30.1
43.6
77.6
32.9
30.2
89.4
43.2
54.6
69.1
38.8
48.5
21.5
30.2
26.6
33.6
45.8
46.9
46.3
74.3
58.9
22.0
31.8
88.4
28.2
35.3
26.1
82.9
79.8
23.0
25.2
28.4
16.1
20.2
–
new structure of 4 was assigned as 3-O-[
(1 ? 2)-b- -xylopyranosyl]-25-O-b- -glucopyranosyl-3b,6
tetrahydroxy-20(R),24(S)-epoxycycloartane.
Additionally, three known cycloartane glycosides namely
3-O-[ -arabinopyranosyl-(1 ? 2)-b- -xylopyranosyl]-3b,6 ,16b,
a-
L-arabinopyranosyl-
D
D
a,16b,25-
108.6
75.0
24.3
25.7
28.5
16.0
20.2
a
-
L
D
a
25-tetrahydroxy-20(R),24(S)-epoxycycloartane (5) (Isaev et al.,
1983a), askendoside C (6) (Isaev et al., 1983b) and askendoside G
(7) (Isaev, 1996) were isolated.
Thus the occurrence of compounds 1–2 characterized by
hydroxylation of ring E and by the esterification of the alcoholic
function at C-23 with hydroxyacetic acid, respectively, along with
compound 3 showing as particular feature the ketalic function at
C-24 is an unusual finding which makes unique the cycloartane
profile of A. campylosema.
AOCOCH3
AOCOCH3
AOCOCH2OH
AOCOCH2OH
172.1
20.7
173.8
61.3
–
–
–
–
–
–
The HRMALDITOF mass spectrum of 3 (m/z 791.4199 [M+Na]+,
calcd for C40H64O14Na, 791.4194) supported a molecular formula
of C40H64O14. The ESIMS mass spectrum showed the sodiated ion
peak at m/z 791 which was assigned to [M+Na]+. The MS/MS of this
ion showed peaks at m/z 659 [M+Naꢀ132]+, corresponding to the
loss of an arabinopyranosyl unit, at m/z 509 [M+Naꢀ132ꢀ150]+,
ascribable to the loss of a xylopyranosyl unit and at m/z 305
[M+Naꢀ486]+, due to the loss of the aglycon moiety.
3. Experimental
3.1. General
Optical rotations were measured on a Rudolph Research Analyt-
ical Autopol IV polarimeter. IR measurements were obtained on a
Bruker IFS-48 spectrometer. NMR experiments were performed
on a Bruker DRX-600 spectrometer (Bruker BioSpinGmBH, Rhein-
stetten, Germany) equipped with a Bruker 5 mm TCI CryoProbe
at 300 K. All 2D-NMR spectra were acquired in CD3OD (99.95%, Sig-
ma Aldrich) and standard pulse sequences and phase cycling were
used for DQF-COSY, HSQC, HMBC and ROESY spectra. The NMR
data were processed using UXNMR software. Exact masses were
measured by a Voyager DE mass spectrometer. Samples were ana-
lyzed by matrix-assisted laser desorption ionization time-of-flight
(MALDITOF) mass spectrometry. A mixture of analyte solution and
As concerning the aglycon moiety, the 1H NMR spectrum
showed signals due to a cyclopropane methylene at d 0.61 and
0.42 (each 1H, d, J = 4.2 Hz), six tertiary methyl groups at d 1.52,
1.36, 1.34, 1.31, 1.30, 1.04 and 0.99, and four methine proton sig-
nals at d 4.35 (ddd, J = 8.0, 8.0, 5.2 Hz), 4.17 (d, J = 6.3 Hz), 3.46
(ddd, J = 9.5, 9.5, 4.5 Hz) and 3.23 (dd, J = 11.3, 4.0 Hz) (Table 3).
Comparison of 1H and 13C NMR data of the aglycon region of 3 with
those of compound 1 revealed that the aglycon of compound 3 dif-
fers from that of 1 by the absence of the signals of C-24 (dH 3.59, d,
J = 6.3 Hz, dC = 90.4) and by the presence of a ketalic function at d
108.6 (Tables 2 and 3). HMBC correlations between the proton sig-
nals at d 1.31 (Me-26), 1.36 (Me-27), 4.17 (H-23) and d 1.44 (H-22)
and the carbon resonance at d 108.6 suggested for the aglycon
a-cyano-4-hydroxycinnamic acid (Sigma) was applied to the
metallic sample plate and dried. Mass calibration was performed
with the ions from ACTH (fragment 18-39) at 2465.1989 Da and
angiotensin III at 931.5154 Da as internal standard. ESIMS analyses
were performed using a ThermoFinnigan LCQ Deca XP Max ion-
trap mass spectrometer equipped with Xcalibur software. GC anal-
ysis was performed on a ThermoFinnigan Trace GC apparatus using
a l-Chirasil-Val column (0.32 mm ꢁ 25 m).
of compound
3
the structure 3b,6a,23a,25-tetrahydroxy-
16b,24;20,24-diepoxycycloartane. An examination of molecular
models indicates that the heterocycles can be fused as the 20R,
24R- and 20S, 24S-configurations. Therefore, establishing the ste-
reochemistry of one of these asymmetric centers defines the con-
figuration of the other chiral atom (Agzamova and Isaev, 1995).
Thus, the new compound 3 was identified as 3-O-[
a-L-arabino-
3.2. Plant material
pyranosyl-(1 ? 2)-b- -xylopyranosyl]-3b,6 ,23 ,25-tetrahydroxy-
D
a
a
20(R),24(R)-16b,24;20,24-diepoxycycloartane. Compounds of this
class are very unusual in the plant kingdom: 16b,24;20,24-diep-
oxycycloartane-type derivatives have been isolated only from
Astragalus campylosema Boiss. subsp. campylosema was col-
lected from Tutak, 1 km from Çobanobası village to Tutak, Ag˘rı,

_
I. Çalıßs et al. / Phytochemistry 69 (2008) 2634–2638
2637
Table 3
1H NMR data (J in Hz) of the aglycon moieties of compounds 1–4 (600 MHz, CD3OD)
Position
1
2
3
4
1
2
3
4
5
6
1.58, 1.25, m
1.57, 1.25, m
1.57, 1.24, m
1.58, 1.25, m
1.95, 1.71, m
3.23, dd (11.3, 4.0)
–
1.39, d (9.5)
3.48, ddd
(9.5, 9.5, 4.5)
1.49, 1.38, m
1.83, dd (11.9, 4.2)
–
1.95, 1.71, m
3.24, dd (11.3, 4.0)
–
1.39, d (9.5)
3.48, ddd
(9.5, 9.5, 4.5)
1.49, 1.38, m
1.83, dd (11.9, 4.2)
–
1.95, 1.71, m
3.23, dd (11.3, 4.0)
–
1.38, d (9.5)
3.47, ddd
(9.5, 9.5, 4.5)
1.45, 1.40, m
1.84, dd (11.9, 4.2)
–
1.95, 1.70, m
3.23, dd (11.3, 4.0)
–
1.37, d (9.5)
3.46, ddd
(9.5, 9.5, 4.5)
1.46, 1.38, m
1.77, dd (11.9, 4.2)
–
7
8
9
10
11
12
13
14
15
–
–
–
–
2.06, 1.25, m
1.73, 1.69, m
–
2.06, 1.28, m
1.81, 1.76, m
–
2.14, 1.22, m
1.70 (2H), m
–
2.06, 1.25, m
1.71, 1.71, m
–
–
–
–
–
1.98, dd (12.7, 8.0)
1.42, dd (12.7, 5.2)
4.68, ddd
(8.0, 8.0, 5.2)
2.34, d (8.0)
1.27, s
0.58, d (4.2)
0.42, d (4.2)
–
2.28, dd (12.7, 8.0)
1.39, dd (12.7, 5.2)
5.48, ddd
(8.0, 8.0, 5.2)
2.59, d (8.0)
1.34, s
0.59, d (4.2)
0.42, d (4.2)
–
1.86, dd (12.7, 8.0)
1.98, dd (12.7, 8.0)
1.45, dd (12.7, 5.2)
4.69, ddd
(8.0, 8.0, 5.2)
2.39, d (8.0)
1.31, s
0.58, d (4.2)
0.41, d (4.2)
–
1.26, s
2.58, m
1.69, m
2.20, m
1.44,a
16
4.35, ddd
(8.0, 8.0, 5.2)
2.58, d (8.0)
1.30, s
0.61, d (4.2)
0.42, d (4.2)
–
17
18
19
20
21
22
1.42, s
1.46, s
1.52, s
3.09, dd (13.7, 9.3)
3.07, dd (13.7, 9.3)
1.61, dd (13.7, 3.4)
5.18, ddd
(9.3, 6.3, 3.4)
3.78, d (6.3)
–
3.03, dd (13.7, 6.3)
1.69,a
1.44,a
23
4.47, ddd
(9.3, 6.3, 3.4)
3.59, d (6.3)
–
4.17, d (6.3)
2.02, m
3.86, dd (8.2, 6.0)
–
24
25
–
–
26
27
28
29
1.28, s
1.30, s
1.33, s
1.05, s
1.16, s
1.18, s
1.32, s
1.04, s
1.31, s
1.36, s
1.34, s
1.04, s
0.99, s
1.26, s
1.41, s
1.33, s
1.04, s
30
1.02, s
1.07, s
1.03, s
AOCOCH3
ACOCH2OH
2.08, s
4.24, d (16.8)
4.11, d (16.8)
a
Overlapped with other signals.
East Anatolia, Turkey in June 23, 2005. Samples of plant material
were identified by one of the co-author, A.A. Dönmez. Voucher
specimen (AAD 12252) has been deposited at the HUB.
ing with CH2Cl2–MeOH–H2O mixtures (from 80:20:1 to 50:50:5) to
give compounds 2 (9 mg) and 4 (6 mg), respectively. Fractions 24–
26 (208 mg) were also applied to a silica gel column (50 g) using
CH2Cl2–MeOH–H2O mixtures (80:20:2, 70:30:3 and 60:40:4) as
eluent to give compounds 6 (28 mg) and 7 (53 mg), respectively.
Fractions 29–30 (55 mg) and 31–33 (45 mg) were rich in com-
pound 5 of which later were further subjected to a normal phase
silica gel column chromatography using CHCl2–MeOH–H2O mix-
ture (80:20:2) to yield the compound 5 (19 mg).
3.3. Extraction and isolation
The air-dried powdered roots (120 g) were macerated with
MeOH (1000 ml) at room temperature 24 h. After filtration of the
MeOH through a funnel, extracted plant material was washed by
MeOH (500 ml). The combined MeOH used in the maceration and
washing was removed by rotary evaporation yielding 8.7 g of crude
extract (yield 7.25%). The crude extract was dissolved in H2O
(30 ml) and applied to VLC using reversed phase material (LiChro-
prep C-18, 26 ꢁ 260 mm), eluting with H2O (A; 200 ml), 20% and
50% MeOH (B and C; each 100 ml) and MeOH (D; 200 ml). Fraction
D eluted with MeOH was rich in cycloartane glycosides (1778 mg).
An aliquot of fraction D (1250 mg) was further applied to MPLC on
reversed phase silica gel (LiChrosorb C-18, 26 ꢁ 260 mm) using
stepwise MeOH–H2O gradient (50–100% MeOH, 35 ml/fraction)
to give 50 fractions. Fractions 3–15 (611 mg) were poor in cyloar-
tane glycosides. Fractions 16–18 were rich in compound 1 (80 mg)
which was further purified on a silica gel column (50 g) eluting
with CH2Cl2–MeOH–H2O mixtures (80:20:1 and 80:20:2, 250 ml
and 550 ml, respectively) to yield pure 1 (13 mg). Fractions 19–
21 (47 mg) consisted of crude compound 3. Final purification was
performed on a silica gel column (30 g) using CH2Cl2–MeOH–H2O
3.4. 3-O-[
a
-L
-Arabinopyranosyl-(1 ? 2)-b-
D-xylopyranosyl]-
3b,6 ,16b,23
a
a
,25-pentahydroxy-20(R),24(S)-epoxycycloartane (1)
Amorphous white solid; C40H66O14; ½a D35
ꢂ
+ 40° (MeOH; c 0.1); IR
KBr
max
m
cmꢀ1: 3472 (>OH), 3035 (cyclopropane ring), 2932 (>CH),
1273 and 1040 (C–O–C); for 13C and 1H NMR data of the sugar por-
tion, see Table 1; for 13C and 1H NMR data of the aglycon moiety,
see Tables 2 and 3, respectively; ESI-MS m/z 793 [M+Na]+; MS/
MS m/z 661 [M+Naꢀ132]+, m/z 511 [M+Naꢀ132ꢀ150]+, m/z 305
[M+Naꢀ488]+; HRMALDITOFMS [M+Na]+ m/z 793.4355 (calcd. for
C40H66O14Na, 793.4350).
3.5. 3-O-[a-L-Arabinopyranosyl-(1 ? 2)-b-D-xylopyranosyl]-16-O-
hydroxyacetoxy-23-O-acetoxy-3b,6
a
,16b,23
a
,25-pentahydroxy-
20(R),24(S)-epoxycycloartane (2)
mixture (80:20:1) to give pure
3
(5 mg). Fractions 22–23
Amorphous white solid; C44H70O17; ½a D35
ꢂ
+ 67° (MeOH; c 0.1); IR
cmꢀ1: 3480 (>OH), 3044 (cyclopropane ring), 2928 (>CH),
KBr
max
(108 mg) was chromatographed on a silica gel column (55 g) elut-
m

_
2638
I. Çalıßs et al. / Phytochemistry 69 (2008) 2634–2638
1734 (C@O), 1277–1035 (CAOAC); for 13C and 1H NMR data of the
sugar portion, see Table 1; for 13C and 1H NMR data of the aglycon
moiety, see Table 2 and 3, respectively; ESI-MS m/z 893 [M+Na]+;
MS/MS m/z 817 [M+Naꢀ76]+, m/z 757 [M+Naꢀ76ꢀ60]+, m/z 685
[M+Naꢀ76ꢀ132]+, m/z 305 [M+Naꢀ76ꢀ512]+; HRMALDITOFMS
[M+Na]+ m/z 893.4519 (calcd. for C44H70O17Na, 893.4511).
12.00 min (
D
-xylose), 8.92 and 9.80 min (
L-arabinose), 14.64 min
(L-glucose) and 11.03 and 12.06 min (L
-xylose).
References
Agzamova, M.A., Isaev, M.I., 1995. Triterpene glycosides and their genins from
Astragalus L. Structure of cycloalpigenin and cycloalpioside. Khim. Prir. Soedin.
5, 700–708.
_
3.6. 3-O-[
3b,6 ,23
diepoxycycloartane (3)
a
-
L
-Arabinopyranosyl-(1 ? 2)-b-
D-xylopyranosyl]-
Bedir, E., Çalıßs, I., Aquino, R., Piacente, S., Pizza, C., 1998a. Cycloartane triterpene
glycosides from the roots of Astragalus brachypterus and Astragalus
a
a,25-tetrahydroxy-20(R),24(R)-16b,24; 20,24-
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_
Bedir, E., Çalıßs, I., Zerbe, O., Sticher, O., 1998b. Cyclocephaloside I:
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Amorphous white solid; C40H64O14; ½a D24
ꢂ
½
a 3D5
+ 20° (MeOH; c
ꢂ
503–505.
_
cmꢀ1: 3470 (>OH), 3040 (cyclopropane ring), 2930
Bedir, E., Çalısß, I., Aquino, R., Piacente, S., Pizza, C., 1999a. Secondary metabolites
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max
0.1); IR
m
from the roots of Astragalus trojanus. J. Nat. Prod. 62, 563–568.
(>CH), 1280-1045 (CAOAC); for 13C and 1H NMR data of the sugar
portion, see Table 1; for 13C and 1H NMR data of the aglycon moi-
ety, see Tables 2 and 3, respectively; ESI-MS m/z 791 [M+Na]+; MS/
MS m/z 659 [M+Naꢀ132]+, m/z 509 [M+Naꢀ132ꢀ150]+, m/z 305
[M+Naꢀ486]+; HRMALDITOFMS [M+Na]+ m/z 791.4199 (calcd. for
C40H64O14Na, 791.4194).
_
Bedir, E., Çalıßs, I., Aquino, R., Piacente, S., Pizza, C., 1999b. Trojanoside H:
a
cycloartane-type secondary glycoside from the aerial parts of Astragalus
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_
Bedir, E., Pugh, N., Çalıßs, I., Pasco, D.S., Khan, I.A., 2000. Immunostimulatory effects
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Çalıßs, I., Sticher, O., 1996. Triterpene saponins from plants of the flora of Turkey. In:
Waller, C.R., Yamasaki, K. (Eds.), Saponins Used in Traditional Medicine
Advances in Experimental Medicine and Biology, vol. 404. Plenum, New York,
pp. 485–500.
3.7. 3-O-[
-glucopyranosyl-3b,6
epoxycycloartane (4)
a
-
L
-Arabinopyranosyl-(1 ? 2)-b-
D-xylopyranosyl]-25-O-b-
_
Çalıßs, I., Yusufog˘lu, H., Zerbe, O., Sticher, O., 1999. Cephalotoside A: a tridesmosidic
D
a,16b,25-tetrahydroxy-20(R),24(S)-
cycloartane type glycoside from Astragalus cephalotes var brevicalyx.
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Çalıßs, I., Yürüker, A., Tasßdemir, D., Wright, A.D., Sticher, O., Luo, Y.D., Pezzuto, J.M.,
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melanophrurius. Planta Med. 63, 183–186.
Amorphous white solid; C46H76O18; ½a D35
ꢂ
+ 24° (MeOH; c 0.1); IR
cmꢀ1: 3475 (>OH), 3047 (cyclopropane ring), 2925 (>CH),
KBr
max
_
_
m
Çalıßs, I., Zor, M., Saracog˘lu, I., Isßimer, A., Rüegger, H., 1996. Four novel cycloartane
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Davis, P.H., 1970. Flora of Turkey and East Aegean Island, vol. 4. University Press,
Edinburgh. pp. 49–254.
1270–1043 (CAOAC); for 13C and 1H NMR data of the sugar por-
tion, see Table 1; for 13C and 1H NMR data of the aglycon moiety,
see Table 2 and 3, respectively; ESI-MS m/z 939 [M+Na]+; MS/MS
m/z 759 [M+Naꢀ180]+, m/z 627 [M+Naꢀ180ꢀ132]+, m/z 477
[M+Naꢀ180ꢀ132ꢀ150]+, m/z 305 [M+Naꢀ180ꢀ454]+; HRMALDI-
TOFMS [M+Na]+ m/z 939.4934 (calcd. for C46H76O18Na, 939.4929).
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
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D from Astragalus
taschkendicus. Khim. Prir. Soedin. 2, 180–185.
Isaev, M.I., Gorovits, M.B., Gorovits, T.T., Abdullaev, N.D., Abubakirov, N.K., 1983b.
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Astragalus taschkendicus. Khim. Prir. Soedin. 2, 173–180.
3.8. Acid hydrolysis
Isaev, M.I., 1996. Triterpene glycosides of Astragalus and their genins LIV.
A solution (1 mg each) of compounds 1 and 4 in 1 N HCl (0.5 ml)
was stirred at 80 °C for 4 h. After cooling, the solution was concen-
trated by blowing with N2. The residue was dissolved in 1-(tri-
methylsilyl)-imidazole and pyridine (0.1 ml), and the solution
was stirred at 60 °C for 5 min. After drying the solution with a
stream of N2, the residue was partitioned between H2O and CH2Cl2
(1 ml, 1:1 v/v). The CH2Cl2 layer was analyzed by GC using an
L-Chirasil-Val column (0.32 mm ꢁ 25 m). Temperatures of the
injector and detector were 200 °C for both. A temperature gradient
system was used for the oven, starting at 100 °C for 1 min and
increasing up to 180 °C at a rate of 5 °C/min. The peaks of the
Askendoside
727.
G from Astragalus taschkendicus. Khim. Prir. Soedin. 5, 723–
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sapogenol XXXV. Chemical constituents of astragali radix, the root of Astragalus
membranaceus Bunge. (2). Astragalosides I, II and IV, acetylastragaloside I and
isoastragalosides I and II. Chem. Pharm. Bull. 31, 698–708.
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_
Özipek, M., Dönmez, A.A., Çalıßs, I., Brun, R., Rüedi, P., Tasdemir, D., 2005.
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hydrolysate of 1 was detected at 8.90 and 9.79 min (
and 10.97 and 12.00 ( -xylose). The peaks of the hydrolysate of 4
were detected at 8.91 and 9.81 min ( -arabinose), 14.74 min
-glucose), 10.96 and 12.02 -xylose). Retention times for
L-arabinose)
D
Sakurai, N., Goto, T., Nagai, M., Inoue, T., Xiao, P., 1990. Studies on the constituents
of Beesia calthaefolia, and Souliea vaginata III. Beesioside IV, a cyclolanostanol
xyloside from the rhizomes of B. calthaefolia and S. vaginata. Heterocycles 30,
897–904.
L
(D
(D
authentic samples after being treated in the same manner with
1-(trimethylsilyl)-imidazole in pyridine were detected at 8.80
_
Yesilada, E., Bedir, E., Çalıßs, I., Takaishi, Y., Ohmoto, Y., 2005. Effects of triterpene
saponins from Astragalus species on in vitro cytokine release. J.
and 9.72 min
(D
-arabinose), 14.71 min (
D
-glucose), 10.98 and
Ethnopharmacol. 96, 71–77.