Cycloartane-type glycosides from Astragalus amblolepis

Article abstract of DOI:10.1016/j.phytochem.2009.03.006

Five cycloartane-type triterpene glycosides were isolated from the methanol extract of the roots of Astragalus amblolepis Fischer along with one known saponin, 3-O-β-D-xylopyranosyl-16-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxy-cycloartane. Stru

Full text of DOI:10.1016/j.phytochem.2009.03.006

Phytochemistry 70 (2009) 628–634
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Cycloartane-type glycosides from Astragalus amblolepis
b
b
c,
*
Emre Polat a, Ozgen Caliskan-Alankus a, , Angela Perrone , Sonia Piacente , Erdal Bedir
*
a
_
Ege University, Faculty of Science, Department of Chemistry, Bornova, 35100 Izmir, Turkey
b Salerno University, Department of Pharmaceutical Sciences, 84084 Fisciano, Salerno, Italy
c
_
Ege University, Faculty of Engineering, Department of Bioengineering, Bornova, 35100 Izmir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Five cycloartane-type triterpene glycosides were isolated from the methanol extract of the roots
of Astragalus amblolepis Fischer along with one known saponin, 3-O-b-D-xylopyranosyl-16-O-b-D-glu-
Received 5 January 2009
Received in revised form 5 March 2009
Available online 6 April 2009
copyranosyl-3b,6
a
,16b,24(S),25-pentahydroxy-cycloartane. Structures of the compounds were
,16b,24(S),25-pentahydroxy-
established as 3-O-b-D-xylopyranosyl-25-O-b-D-glucopyranosyl-3b,6
a
cycloartane, 3-O-[b-D-glucuronopyranosyl-(1 ? 2)-b-D-xylopyranosyl]-25-O-b-D-glucopyranosyl-3b,6
16b,24(S),25-pentahydroxy-cycloartane, 3-O-b-D-xylopyranosyl-24,25-di-O-b-D-glucopyranosyl-3b,6
a
a
,
,
Keywords:
Astragalus amblolepis
Leguminosae
Cycloartane
Glucuronic acid
Saponin
16b,24(S),25-pentahydroxy-cycloartane, 6-O-
,16b,24(S),25-pentahydroxy-cycloartane, 6-O-
3b,6 ,16b,24(S),25-pentahydroxy-cycloartane by using 1D and 2D-NMR techniques and mass spec-
a
-L-rhamnopyranosyl-16,24-di-O-b-D-glucopyranosyl-3b,
6
a
a
-L-rhamnopyranosyl-16,25-di-O-b-D-glucopyranosyl-
a
trometry. To the best of our knowledge, the glucuronic acid moiety in cycloartanes is reported for
the first time.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
glycosidic forms, pterocarpans free and as glycosides and organic
acid derivatives (Rios and Waterman, 1997).
The genus Astragalus belonging to the Leguminosae family is
widely distributed throughout the temperate regions of the world,
located principally in Europe, Asia and North America. About 2000
species have been described, 372 of them in North America and
133 in Europe (Rios and Waterman, 1997; Davis, 1982). In the flora
of Turkey, this genus is represented by ꢀ380 species, which are
listed under several sections (Davis, 1970).
The roots of Astragalus are used in traditional medicine as an
antiperspirant, diuretic and tonic drug. It has also been used in
the treatment of diabetes mellitus, nephiritis, leukemia and uterine
cancer (Tang and Eisenbrand, 1992). In the district of Anatolia, lo-
cated in South Eastern Turkey, an aqueous extract of the roots of
Astragalus is traditionally used against leukemia and for its wound
healing properties.
The genus Astragalus appears highly uniform from chemical
point of view, with two kinds of pharmacologically active princi-
ples and three different kinds of toxic compounds. In the former
group, the polysaccharides and the saponins stand out, and in
the second, the indolizidine alkaloids, the nitro compounds and
3-nitropropyl glucosides, and the selenium compounds. There are
other interesting compounds, such as flavonoids in free and
Astragalus polysaccharides are known to have anticancer and
immune enhancing properties in both in vitro and in vivo experi-
ments (Simee and Verbiscar, 1995; Yang and Zhao, 1998; Liu
et al., 1994).
Chemical studies on Astragalus saponins have indicated the
presence of cycloartane-type triterpenoid glycosides which were
found to exert biological activities, e.g. antiinflammatory, analge-
sic, diuretic, hypotensive and sedative effects (Isaev et al., 1989;
Çalıßs et al., 1997).
Earlier investigations on Turkish Astragalus species resulted in
the isolation of a series of cycloartane-type triterpenoidal saponins
(Bedir et al., 1998, 1999, 2001a; Çalıßs et al., 1999, 2008; Tabanca
et al., 2005). Continuing our studies on the constituents of Astrag-
alus species, we investigated the roots of Astragalus amblolepis
Fischer. This paper describes the isolation and structure elucida-
tion of five new cycloartane-type glycosides.
2. Results and discussion
The HRMALDITOF mass spectrum of 3-O-b-D-xylopyranosyl-
25-O-b-D-glucopyranosyl-3b,6
a,16b,24(S),25-pentahydroxy-cycl-
oartane (1) (m/z 809.4669 [M+Na]+, calc. for C41H70O14Na,
809.4663) supported a molecular formula of C41H70O14. The ESI–
MS mass spectrum showed the major ion peak at m/z 809.4 which
was assigned to [M+Na]+. The MS/MS of this ion showed peaks at
m/z 629.3 [M+Naꢁ180]+, corresponding to the loss of an hexose
* Corresponding authors. Tel./fax: +90 232 3888264 (O. Caliskan-Alankus),
tel./fax: +90 232 388 4955 (E. Bedir).
E-mail addresses: ozgen.alankus.caliska@ege.edu.tr (O. Caliskan-Alankus), erdal.
bedir@ege.edu.tr (E. Bedir).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.03.006

E. Polat et al. / Phytochemistry 70 (2009) 628–634
629
Table 1
1H NMR data (J in Hz) of the aglycon moieties of compounds 1–6 (600 MHz, d ppm, in CD3OD)a.
1
2
3
4
5
6
H2-1
H2-2
H-3
H-5
H-6
H2-7
H-8
H2-11
H2-12
H2-15
1.56, 1.26, m
1.97, 1.69, m
3.24, dd (11.3, 4.0)
1.40, d (9.5)
3.48, ddd (9.5, 9.5, 4.5)
1.50, 1.37, m
1.81, dd (11.9, 4.2)
2.00, 1.22, m
1.73, 1.68, m
2.05, dd (12.7, 8.0)
1.42, dd (12.7, 5.2)
4.47, ddd (8.0, 8.0, 5.2)
1.73, dd (9.9, 8.0)
1.21, s
1.56, 1.26, m
1.97, 1.69, m
3.24, dd (11.3, 4.0)
1.40, d (9.5)
3.48, ddd (9.5, 9.5,4.5)
1.50, 1.37, m
1.81, dd (11.9, 4.2)
2.02, 1.22, m
1.73, 1.68, m
2.08, dd (12.7, 8.0)
2.06, dd (12.7, 5.2)
4.27, ddd (8.0, 8.0, 5.2)
1.84, dd (9.9, 8.0)
1.21, s
1.56, 1.26, m
1.97, 1.69, m
3.25, dd (11.3, 4.0)
1.39, d (9.5)
3.46, ddd (9.5, 9.5, 4.5)
1.48, 1.38, m
1.82, dd (11.9, 4.2)
2.00, 1.23, m
1.71, 1.68, m
2.05, dd (12.7, 8.0)
1.42, dd (12.7, 5.2)
4.48, ddd (8.0, 8.0, 5.2)
1.72, dd (9.9, 8.0)
1.20, s
1.56, 1.26, m
1.97, 1.69, m
3.24, dd (11.3, 4.0)
1.40, d (9.5)
3.48, ddd (9.5, 9.5, 4.5)
1.50, 1.37, m
1.81, dd (11.9, 4.2)
2.02, 1.22, m
1.73, 1.68, m
2.04, dd (12.7, 8.0)
1.43, dd (12.7, 5.2)
4.46, ddd (8.0, 8.0, 5.2)
1.75, dd (9.9, 8.0)
1.21, s
1.59, 1.29, m
1.72, 1.65, m
3.23, dd (11.3, 4.0)
1.53, d (9.5)
3.42, ddd (9.5, 9.5, 4.5)
1.85, 1.39, m
1.79, dd (11.9, 4.2)
2.00, 1.24, m
1.71, 1.65, m
2.07, dd (12.7, 8.0)
1.80, dd (12.7, 5.2)
4.27, ddd (8.0, 8.0, 5.2)
1.89, dd (9.9, 8.0)
1.20, s
1.59, 1.29, m
1.72, 1.65, m
3.23, dd (11.3, 4.0)
1.53, d (9.5)
3.42, ddd (9.5, 9.5, 4.5)
1.85, 1.39, m
1.79, dd (11.9, 4.2)
2.00, 1.24, m
1.71, 1.65, m
2.07, dd (12.7, 8.0)
1.80, dd (12.7, 5.2)
4.27, ddd (8.0, 8.0, 5.2)
1.89, dd (9.9, 8.0)
1.20, s
H-16
H-17
H3-18
H2-19
0.55, d (4.2)
0.41, d (4.2)
1.90, m
0.55, d (4.2)
0.41, d (4.2)
2.10, m
0.54, d (4.2)
0.40, d (4.2)
1.90, m
0.55, d (4.2)
0.41, d (4.2)
1.84, m
0.54, d (4.2)
0.40, d (4.2)
2.09, m
0.54, d (4.2)
0.40, d (4.2)
1.92, m
H-20
H3-21
H2-22
H2-23
H-24
H3-26
H3-27
H3-28
H3-29
H3-30
0.97, d (6.5)
1.81, 1.24, m
1.59, 1.51, m
3.61, dd (2.4, 10.5)
1.29, s
1.24, s
1.32, s
1.05, s
0.98, s
0.96, d (6.5)
1.81, 1.24, m
1.67, 1.40, m
3.41, dd (2.4, 10.5)
1.18, s
1.18, s
1.32, s
1.05, s
0.98, s
0.97, d (6.5)
1.81, 1.24, m
1.59, 1.51, m
3.61, dd (2.4, 10.5)
1.29, s
1.25, s
1.31, s
1.02, s
0.99, s
0.99, d (6.5)
1.81, 1.24, m
1.85, 1.79, m
3.77, dd (2.4, 10.5)
1.37, s
1.32, s
1.32, s
1.05, s
0.98, s
0.96, d (6.5)
1.69, 1.60, m
1.60 (2H), m
3.58, dd (2.4, 10.5)
1.21, s
1.19, s
1.13, s
0.98, s
0.98, s
0.96, d (6.5)
2.00, 0.89, m
1.83, 1.19, m
3.41, dd (2.4, 10.5)
1.25, s
1.27, s
1.13, s
0.98, s
0.98, s
a
Assignments confirmed by 1D-TOCSY, DQF-COSY, HSQC and HMBC experiments.
unit and at m/z 479.0 [M+Naꢁ180ꢁ150]+, due to the loss of a pen-
tose unit. Taking into account the results of our comprehensive 1H
and 13C NMR studies, the main features of a cyclopropane-type tri-
terpene possessing an acyclic side chain were evident for com-
pound 1: characteristic signals due to cyclopropane–methylene
protons as an AX system (d 0.41, 0.55, JAX = 4.2 Hz, H2-19), six ter-
tiary methyl groups (d 1.05, 1.21, 0.98, 1.32, 1.24, 1.29; respec-
tively, H3-29, H3-18, H3-30, H3-28, H3-27, H3-26) and a secondary
methyl group (d 0.97, d, J = 6.5, H3-21) (Bedir et al., 2001b) (Table
1). Additionally, the resonances for two anomeric protons were ob-
served at d 4.30 (d, J = 7.5 Hz) and 4.55 (d, J = 7.5 Hz), indicative of
the presence of two b-linked sugar units (Table 2). Thus, 1 was con-
sidered to be a cycloartane-type triterpene diglycoside.
with cyclocanthogenin (Isaev et al., 1989). Full assignment of the
1H and 13C signals of the aglycon part of 1, accomplished by
DQF-COSY and HSQC spectra, showed glycosylation shifts for C-3
(d 89.6) and C-25 (d 81.4). All connectivities within 1 were also
confirmed by HMBC spectrum, which showed a long-range corre-
lation between the anomeric proton signal at d 4.30 (d, J = 7.5 Hz)
and the carbon resonance at d 89.6 (C-3), while the anomeric pro-
ton at d 4.55 (d, J = 7.5 Hz) exhibited a long-range correlation with
the carbon resonance at d 81.4 (C-25), confirming the bidesmosidic
character of 1. Furthermore the following stereochemical data con-
firmed the presence of cyclocanthogenol as aglycon of compound
1. The relative configurations of the oxygenated carbon atoms were
determined from the magnitude of the vicinal proton–proton cou-
pling constants: C-3-(b-OH) (d 3.24, dd, J = 11.3, 4.0 Hz, Hax-3), C-6-
The 13C NMR spectrum contained 41 resonances (Table 3); 30 of
them, attributed to the sapogenol moiety, were in good agreement
(a-OH) (d 3.48, ddd, J = 9.5, 9.5, 4.5 Hz, Hax-6), C-16-(b-OH) (d 4.47,
Table 2
1H NMR data (J in Hz) of the sugar moieties of compounds 1–6 (600 MHz, d ppm, in CD3OD)a.
1
2
3
4
5
6
H-10
H-20
H-30
H-40
H-50
4.30 d (7.5)
3.22 dd (9.2, 7.5)
3.31 dd (9.2)
3.49 m
3.84 dd (11.7, 5.2)
3.20 t (11.7)
–
4.31 d (7.5)
3.22 dd (9.2, 7.5)
3.34, dd (9.2)
3.49 m
3.85 dd (11.7, 5.2)
3.21 t (11.7)
–
4.56 d (7.5)
3.65 dd (9.2, 7.5)
3.58, dd (9.2)
3.54 m
3.91 dd (11.7, 5.2)
3.24 t (11.7)
–
4.30 d (7.5)
4.78 d (1.2)
3.86 dd (1.2, 3.2)
3.62 dd (3.2, 9.3)
3.42 t (9.3)
4.78 d (1.2)
3.22 dd (9.2, 7.5)
3.31, dd (9.2)
3.49 m
3.84 dd (11.7, 5.2)
3.20 t (11.7)
3.86 dd (1.2, 3.2)
3.62 dd (3.2, 9.3)
3.42 t (9.3)
3.72 m
3.72 m
H-60
–
1.29 s
1.29 s
H-100
H-200
H-300
H-400
H-500
H-600
4.55 d (7.5)
3.20 dd (7.5, 9.0)
3.39 dd (9.0, 9.0)
3.30 dd (9.0, 9.0)
3.30 m
3.67 dd (4.5, 12.0)
3.86 dd (3.5, 12.0)
–
–
–
–
–
–
4.24 d (7.5)
3.22 dd (7.5, 9.0)
3.38 dd (9.0, 9.0)
3.32 dd (9.0, 9.0)
3.25 m
3.71 dd (4.5, 12.0)
3.85 dd (3.5, 12.0)
–
–
–
–
–
–
4.66 d (7.5)
3.35 dd (7.5, 9.0)
3.46 dd (9.0, 9.0)
3.49 dd (9.0, 9.0)
3.64 d 9.0)
4.45 d (7.5)
4.32 d (7.5)
4.30 d (7.5)
3.25 dd (7.5, 9.0)
3.42 dd (9.0, 9.0)
3.33 dd (9.0, 9.0)
3.33 m
3.69 dd (4.5, 12.0)
3.89 dd (3.5, 12.0)
4.60 d (7.5)
3.20 dd (7.5, 9.0)
3.39 dd (9.0, 9.0)
3.30 dd (9.0, 9.0)
3.30 m
3.67 dd (4.5, 12.0)
3.86 dd (3.5, 12.0)
3.18 dd (7.5, 9.0)
3.38 dd (9.0, 9.0)
3.39 dd (9.0, 9.0)
3.27 m
3.69 dd (4.5, 12.0)
3.89 dd (3.5, 12.0)
4.45 d (7.5)
3.25 dd (7.5, 9.0)
3.42 dd (9.0, 9.0)
3.33 dd (9.0, 9.0)
3.33 m
3.69 dd (4.5, 12.0)
3.89 dd (3.5, 12.0)
3.15 dd (7.5, 9.0)
3.39 dd (9.0, 9.0)
3.37 dd (9.0, 9.0)
3.24 m
3.69 dd (4.5, 12.0)
3.89 dd (3.5, 12.0)
4.55 d (7.5)
3.20 dd (7.5, 9.0)
3.39 dd (9.0, 9.0)
3.30 dd (9.0, 9.0)
3.30 m
3.67 dd (4.5, 12.0)
3.86 dd (3.5, 12.0)
–
H-1000
H-2000
H-3000
H-4000
H-5000
H-6000
4.55 d (7.5)
3.20 dd (7.5, 9.0)
3.39 dd (9.0, 9.0)
3.30 dd (9.0, 9.0)
3.30 m
3.67 dd (4.5, 12.0)
3.86 dd (3.5, 12.0)
a
Assignments confirmed by 1D-TOCSY, DQF-COSY, HSQC and HMBC experiments.

630
E. Polat et al. / Phytochemistry 70 (2009) 628–634
Table 3
being a cycloartane-type triterpene diglycoside. Full assignments
13C NMR data of compounds 1–6 (150 MHz, d ppm, in CD3OD)a.
of 1H and 13C NMR data of the aglycon of 2 were deduced from
its HSQC and HMBC spectra. All 1D and 2D NMR data of 2 were
superimposable with those of the known compound 3-O-b-D-xylo-
Position
1
2
3
4
5
6
1
33.1
30.3
89.6
42.7
54.5
69.4
38.7
48.7
21.6
29.8
26.7
33.5
46.1
47.5
48.6
73.2
57.9
18.9
31.5
29.8
17.9
33.6
28.4
76.7
81.4
22.9
22.6
28.3
16.3
20.2
107.4
75.1
77.7
70.8
66.3
–
97.8
75.0
78.0
71.2
77.8
62.2
–
33.1
30.3
89.6
42.7
54.7
69.4
38.7
48.7
21.6
29.8
26.7
33.5
46.1
47.5
48.4
83.4
58.1
18.9
31.5
29.8
17.9
33.7
28.6
78.6
73.6
24.9
24.9
28.3
16.3
20.1
107.2
75.0
77.7
70.8
66.2
–
106.2
75.0
78.2
71.2
77.5
62.4
–
33.1
30.3
89.8
43.1
54.6
69.4
38.7
48.7
22.3
30.0
26.6
34.0
46.8
47.6
48.4
73.0
58.2
19.0
31.5
29.8
18.7
33.6
28.4
76.8
81.3
23.0
22.6
28.7
16.5
20.2
105.0
81.4
76.4
71.0
66.3
–
104.3
75.5
77.2
73.6
77.3
177.0
97.7
75.0
78.0
71.2
77.8
62.2
33.1
30.3
89.6
42.7
54.5
69.4
38.7
48.7
21.6
29.8
26.7
33.5
46.1
47.5
49.0
72.7
57.6
18.9
31.5
31.6
18.0
34.1
28.4
85.2
81.0
25.3
22.7
28.3
16.3
20.1
107.1
75.1
77.7
70.8
66.3
–
104.8
75.4
77.8
71.3
77.8
62.4
98.4
75.0
78.0
71.2
77.8
62.2
33.2
30.9
79.0
42.5
53.1
81.4
35.6
48.0
21.7
29.8
26.5
33.3
46.2
48.0
49.0
83.7
57.1
18.9
31.3
31.4
17.9
34.3
29.4
90.4
73.7
24.7
26.5
28.9
16.2
20.1
103.9
72.5
72.3
73.6
69.9
17.7
106.8
75.5
78.0
71.2
77.6
62.4
104.8
75.4
77.8
71.3
77.8
62.4
33.2
30.9
79.0
42.5
53.1
81.4
35.6
48.0
21.7
29.8
26.5
33.3
46.2
48.0
49.0
83.7
57.1
18.9
31.3
31.4
17.9
34.3
29.4
79.1
81.6
20.9
23.2
28.9
16.2
20.1
103.9
72.5
72.3
73.6
69.9
17.7
106.0
75.5
77.8
71.2
77.2
62.4
98.4
75.0
78.0
71.2
77.8
62.2
pyranosyl,16-O-b-D-glucopyranosyl-3b,6a,16b,24(S),25-penta-
2
3
4
5
6
7
8
9
hydroxy-cycloartane, isolated previously from Tragacantha
stipulosa Boviss (Karimov et al., 1998) (Fig. 1, Tables 1–3).
The molecular formula of 3-O-[b-D-glucuronopyranosyl-
(1 ? 2)-b-D-xylopyranosyl]-25-O-b-D-glucopyranosyl-3b,6
24(S),25-pentahydroxy-cycloartane (3) and 3-O-b-D-xylopyrano-
syl-24,25-di-O-b-D-glucopyranosyl-3b,6 ,16b,24(S),25-pentahydr-
a,16b,
a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
10
oxy-cycloartane (4) was unequivocally established to be C47H78O20
and C47H80O19 by HRMALDITOFMS analysis (m/z 985.4979
[M+Na]+, calc. for C47H78O20Na, 985.4984, and m/z 971.5199
[M+Na]+, calc. for C47H80O19Na, 971.5192), respectively.
The NMR spectra of compounds 3 and 4 were also characteris-
tic of cyclocanthogenol type glycosides (Tables 1–3). Furthermore,
the 1H NMR spectrum of 3 clearly showed three anomeric proton
doublets at d 4.66 (J = 7.5 Hz), 4.56 (J = 7.5 Hz) and 4.55 (J = 7.5 Hz)
in the downfield region, indicative of b-linked sugar units. The 13C
NMR spectrum contained 47 resonances; 30 of them attributed to
cyclocanthogenol moiety. Full assignment of the 1H and 13C sig-
nals of the aglycon part of 3 showed glycosylation shifts for C-3
(d 89.8) and C-25 (d 81.3). These results suggested that 3 was also
a bidesmosidic saponin with sugar residues linked to C-3 and C-
25 of cyclocanthogenol. A combination of 1D-TOCSY and HSQC
experiments allowed us to unambiguously determine all sugar
signals and to identify the sugar moieties as consisting of b-xylo-
pyranosyl, b-glucopyranosyl and b-glucuronic acid units,
respectively.
All connectivities, including the sites of attachment of sugar
moieties on the aglycon of 3, were determined by HMBC experi-
ment (Fig. 2). In the HMBC spectrum, the anomeric proton signal
at d 4.56 (H-10), assigned to the b-xylopyranose, showed long-
range correlation with the carbon resonance at d 89.8 (C-3). The
second anomeric proton signal at d 4.66 (H-100), assigned to the
b-glucuronic acid, showed long-range correlation with the carbon
resonance at d 81.4 (C-20). The third anomeric proton signal at d
4.55 (H-1000), assigned to the b-glucopyranose, showed long-range
correlation with the carbon resonance at d 81.3 (C-25). The D con-
figuration of glucose, xylose and glucuronic acid units was deter-
mined by acid hydrolysis followed by GC analysis.
20
30
40
50
60
100
200
300
400
500
600
1000
2000
3000
4000
5000
6000
–
–
–
–
–
–
–
–
–
–
Thus the structure of 3 was elucidated as 3-O-[b-D-glucurono-
pyranosyl-(1 ? 2)-b-D-xylopyranosyl]-25-O-b-D-glucopyranosyl-
a
Assignments confirmed by DQF-COSY, HSQC, and HMBC experiments.
3b,6a,16b,24(S),25-pentahydroxy-cycloartane. This compound (3)
represents the first report of a cycloartane-type compound pos-
sessing glucuronic acid residue in plant kingdom (Fig. 1). Since
most of the Astragalus members have not been studied phytochem-
ically, it is too premature to draw a conclusion from chemotaxo-
nomic point of view especially for Astragalus genus. Thus further
studies are required to clarify chemotaxonomic significance of
the presence of glucuronic acid moiety attached to cycloartanes
in Astragalus species.
ddd, J = 8.0, 8.0, 5.2 Hz, Hax-16). Moreover, 13C-NMR data for C-24
was comparable to those reported for analogous compounds hav-
ing a 24(S) configuration (Bedir et al., 2000; Fadeev et al., 1987;
Hirotani et al., 1994).
As concerning the sugar moiety, examination of the remaining
signals allowed us to identify one b-xylopyranosyl unit and one
b-glucopyranosyl unit (Agrawal et al., 1985; Bedir et al., 2001b).
The configuration of glucose and xylose units was established as
D after hydrolysis of 1 with 1 N HCl, trimethylsilation and determi-
nation of retention time by GC.
The 1H NMR spectrum of 4 showed three anomeric proton res-
onances at d 4.30 (d, J = 7.5 Hz), 4.45 (d, J = 7.5 Hz), and 4.60 (d,
J = 7.5 Hz) correlated by HSQC to the resonances at d 107.1,
104.8 and 98.4 (Tables 2 and 3). The three terminal sugar units
were identified as a b-D-xylopyranose and two b-D-glucopyran-
oses, respectively, using a combination of 1D-TOCSY and HSQC
spectra.
Consequently, the structure of 1 was established as 3-O-b-D-
xylopyranosyl-25-O-b-D-glucopyranosyl-3b,6a,16b,24(S),25-pen-
tahydroxy-cycloartane (Fig. 1).
The 13C-NMR resonances arising from the sapogenol moiety
were similar to those of 3, except for the signal assigned to C-24
(d 85.2) exhibiting a significant glycosidation shift. These results
suggested another tridesmosidic structure in which the three sugar
units were attached to the hydroxyl groups at C-3, C-24 and C-25.
The glycosidation sites were established by the HMBC experiment
The HRMALDITOF mass spectrum of 3-O-b-D-xylopyranosyl,16-
O-b-D-glucopyranosyl-3b,6a,16b,24(S),25-pentahydroxy-cycl-
oartane (2) showed a major ion peak at m/z 809.4665 [M+Na]+
ascribable to molecular formula C41H70O14 (calc. for C41H70O14Na,
809.4663). The NMR spectral data of 2 were consistent with 1

E. Polat et al. / Phytochemistry 70 (2009) 628–634
631
21
OR4
22
18
26
20
24
25
23
12
17
15
13
14
19
5
11
OR5
16
OR3
27
1
4
9
2
3
10
8
30
7
6
R1O
29
28
OR2
R1
R2
H
R3
R5
R4
O
O
H
H
HO
HO
HO
1
2
OH
HO
HO
OH
O
O
H
HO
HO
HO
H
H
OH
HO
HO
OH
O
O
HO
HO
O
H
H
H
H
H
HO
HO
3
HO
OH
O
HOOC
HO
HO
HO
OH
O
O
O
HO
HO
HO
4
5
HO
HO
OH
OH
HO
HO
OH
O
O
O
O
HO
HO
H
H
HO
HO
HO
HO
HO
HO
H
OH
OH
HO
HO
OH
O
O
6
HO
H
HO
HO
HO
HO
OH
OH
HO
OH
Fig. 1. Structures of 1–6.
OH
OH
OH
O
O
OH
HO
OH
O
O
O
HO
HO
OH
HOOC
O
HO
HO
OH
Fig. 2. Key HMBC of 3.
which showed correlations between H-3 (d 3.24) and C-10 (d 107.1)
of the b-D-xylopyranosyl unit, H-24 (d 3.77) and C-100 (d 104.8) of
the first b-D-glucopyranosyl and between H-1000 (d 4.60) and C-25
(d 81.0).
The HRMALDITOF mass spectrum of 6-O-
syl-16,24-di-O-b-D-glucopyranosyl-3b,6
oxy-cycloartane (5) (m/z 985.5350 [M+Na]+, calc. for
C48H82O19Na, 985.5348) supported molecular formula of
a
-L-rhamnopyrano-
a
,16b,24(S),25-pentahydr-
a
On the basis of these evidence, the structure of compound 4 was
established as 3-O-b-D-xylopyranosyl-24,25-di-O-b-D-glucopyran-
osyl-3b,6a,16b,24(S),25-pentahydroxy-cycloartane (Fig. 1).
C48H82O19. The ESI–MS mass spectrum showed the major ion
peak at m/z 985.3 which was assigned to [M+Na]+. The MS/
MS of this ion showed peaks at m/z 805.3 [M+Naꢁ180]+, cor-

632
E. Polat et al. / Phytochemistry 70 (2009) 628–634
responding to the loss of an hexose unit and at m/z 641.4
[M+Naꢁ180ꢁ164]+, ascribable to the loss of a 6-deoxyhexose.
The 1H NMR spectrum of aglycon moiety of 5 displayed the
typical signals of cycloartanes having acyclic side chain: six
3. Experimental
3.1. General procedures
tertiary methyl groups, from signals at
d 0.98, 1.20, 0.98,
Optical rotations were measured on a JASCO DIP 1000 polarim-
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
NMR spectra were acquired in CD3OD (99.95%, Sigma–Aldrich)
and standard pulse sequences and phase cycling were used for
DQF-COSY, HSQC and HMBC spectra. The NMR data were pro-
cessed using UXNMR software. Exact masses were measured by a
Voyager DE mass spectrometer. Samples were analysed by ma-
trix-assisted laser desorption ionization time-of-flight (MALDITOF)
1.13, 1.21, 1.19, and a secondary methyl group from signal
at d 0.96 (d, J = 6.5 Hz, H3-21), as well as the cyclopropane-
methylene protons at d 0.40, 0.54 (JAX = 4.2 Hz, H2-19) (Table
1). In addition, the resonances of three anomeric protons,
indicative of the presence of one
a- linked and two b-linked
sugar moieties, were observed in the downfield region (d
4.78, d, J = 1.2 Hz, H-10; d 4.32, d, J = 7.5 Hz, H-100; d 4.45, d,
J = 7.5 Hz, H-1000) (Table 2).
Combination of 1D-TOCSY and HSQC experiments allowed the
sequential assignments of all proton resonances of each sugar res-
idue, starting from anomeric protons. Furthermore, on the basis of
chemical shifts, the multiplicity of the signal and the coupling con-
mass spectrometry. A mixture of analyte solution and
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. ESI–MS analyses were per-
formed using a ThermoFinnigan LCQ Deca XP Max iontrap mass
spectrometer equipped with Xcalibur software. GC analysis was
performed on a Termo Finnigan Trace GC apparatus using a l-
Chirasil-Val column (0.32 mm ꢂ 25 m). Column chromatography
stants, the three sugar residues were identified as a-rhamnopyra-
nose and two b-glucopyranoses. The D configuration of glucose
units and the L configuration of rhamnose unit were determined
by acid hydrolysis followed by GC analysis.
The remaining 1H and 13C resonances of 5 arising from the
sapogenol moiety were evident for the presence of cyclocanthog-
enol except for the signals ascribable to linkage sites exhibiting
typical glycosidation shifts. Key correlation peaks observed in
the HMBC spectrum of 5 between H-10 of the rhamnosyl at d
4.78 (d, J = 1.2, Hz) and C-6 (d 81.4) of the aglycon, between H-
100 of the glucosyl at d 4.32 (d, J = 7.5 Hz and C-16 (d 83.7), and be-
tween H-1000 of the second glucosyl unit at d 4.45 (d, J = 7.5 Hz)
and C-24 (d 90.4), revealed the locations of glycosidic linkages
to be C-6, C-16 and C-24 indicating the tridesmosidic nature of
compound 5.
was carried out on Silica gel (JT Baker, 40
(Amersham Biosciences, 17-0090-02) and RP (C-18, 40
l
m), Sephadex LH-20
m)
l
(Merck). TLC analyses were carried out on Silica gel 60 F254 (Merck)
and RP-18 F254s (Merck) plates. Compounds were detected by UV
and 20% H2SO4/water spraying reagent followed by heating at
110 °C for 1–2 min.
3.2. Plant material
The molecular formula of 6-O-
a-L-rhamnopyranosyl-16,25-di-
O-b-D-glucopyranosyl-3b,6 ,16b,24(S),25-pentahydroxy-cycloar-
a
A. amblolepis was collected from Ersele Village, Pütürge-
Malatya, East Anatolia, in October 2006 and identified by Serdar
G. Senol (Deparment of Biology, Faculty of Sciences, Ege University,
Izmir, Turkey). A voucher specimen was deposited in the Herbar-
ium of Ege University, Izmir, Turkey (EGE 32574).
tane (6) was determined to be C48H82O19 by HRMALDITOFMS
analysis (m/z 985.5341 [M+Na]+, calc. for C48H82O19Na,
985.5348). In the positive ESI–MS spectrum compound 6 showed
a major ion peak at m/z 985.4 [M+Na]+ and a significant frag-
ment in MS/MS analysis at m/z 805.3 [M+Naꢁ180]+, ascribable
to the loss of an hexose unit. The MS3 fragmentation showed a
peak at m/z 641.3 [M+Naꢁ180ꢁ164]+ corresponding to the loss
of a 6-deoxyhexose unit. Full assignments of the proton and car-
bon signals of the aglycon and sugar parts of 6 in comparison
with those of 5 showed their considerable structural similarity
(Tables 1 and 2). The difference consisted only in the position
of one of the glucose moieties. The sapogenol moiety showed
an unambiguous glycosidation shift at C-25 (d 81.4), while C-
24 displayed no downfield shift due to the sugar attachment (d
79.1). Furthermore, the HMBC experiment showed key correla-
tions peaks between the anomeric proton of rhamnosyl unit (d
4.78, d, J = 1.2 Hz, H-10) and C-6 (d 81.4), between the anomeric
proton of the first glucose moiety (d 4.27, d, J = 7.5 Hz, H-100)
and C-16 (d 83.7), and H-1000 (d 4.55, d, J = 7.5 Hz) of the second
glucose residue and C-25 (d 81.6). Thus, compound 6 displayed
the glycosidation at C-25 instead of the glycosidation at C-24
as for compound 5.
3.3. Extraction and isolation
Air-dried and powdered roots of A. amblolepis (600 g) were ex-
tracted with MeOH (3 ꢂ 3.5 l) at 60 °C. After filtration, the solvent
was removed by rotary evaporation yielding 36 g of extract. The
MeOH extract was dissolved in H2O (400 ml), and successively par-
titioned with n-hexane (3 ꢂ 100 ml), CH2Cl2 (4 ꢂ 100 ml), and n-
BuOH saturated with H2O (6 ꢂ 100 ml). The n-BuOH extract (5 g)
was subjected to vacuum liquid chromatography (VLC) using re-
versed-phase material (Lichroprep RP-18, 25-40 lm, 120 g)
employing H2O (850 ml), H2O–MeOH (8:2, 200 ml; 6:4, 400 ml;
4:6, 1600 ml) and MeOH (800 ml) to give ten main fractions (1–
10). Fractions 7, 8 and 9 eluted with H2O:MeOH (4:6) were rich
in saponins. Fraction 7 (615.2 mg) was subjected to high perfor-
mance flash chromatography (HPFC, Biotage, Inc., A Dyax Corp.
Company; Biotage SI 40 M column), and eluted with CHCl3–MeOH
mixtures [80% CHCl3, 5 column volume (CV); 80–70% CHCl3, 5 CV;
70% CHCl3, 4 CV; 70–60% CHCl3, 4 CV; 50% CHCl3, 3 CV] and MeOH
(4 CV) to yield 510 fractions. Fractions 374-488 and the first MeOH
fraction (160.6 mg) were combined and applied to a reversed-
Consequently, the structure of 5 and 6 was established as 6-O-
a
-L-rhamnopyranosyl-16,24-di-O-b-D-glucopyranosyl-3b,6
24(S),25-pentahydroxy-cycloartane and 6-O- -L-rhamnopyrano-
syl-16,25-di-O-b-D-glucopyranosyl-3b,6 ,16b,24(S),25-pentahydr-
a,16b,
a
a
phase column (Lichroprep RP-18, 25–40 lm, 50 g) using MeOH–
oxy-cycloartane, respectively (Fig. 1). Cycloartane glycosides with
no sugar residue at C-3 position such as compounds 5 and 6 are
rather unusual in nature. Moreover, as far as we have ascertained,
a rhamnosyl unit at C-6 position is reported for the first time in
cyclocanthogenol skeleton which is one of the most common agly-
cons in Astragalus genus together with cycloastragenol.
H2O (5:5, 400 ml; 6:4, 300 ml) to give 1 (4.1 mg). Fractions
287–310 (81.4 mg) were combined and fractionated over an open
column chromatography using Si gel (50 g) as stationary phase.
Elution was performed with CHCl3–MeOH–H2O mixtures
(80:20:2, 300 ml; 70:30:3, 1700 ml; 61:32:7, 400 ml) to afford 2

E. Polat et al. / Phytochemistry 70 (2009) 628–634
633
(19.8 mg). Fractions 8 and 9 (860.2 mg) from VLC were combined
and subjected to Si gel (200 g) column chromatography. Elution
was carried out with CHCl3-MeOH–H2O (85:15:0.5, 400 ml),
CHCl3-MeOH (80:20, 400 ml) and CHCl3–MeOH–H2O (80:20:1,
500 ml) yielding 3 (3.9 mg) and 4 (21.8 mg). Fractions 894–1110
(135 mg) were applied to column chromatography using re-
versed-phase material (Lichroprep RP-18, 25–40 lm, 40 g)
employing MeOH:H2O (6:4, 350 ml) to give 5 (5.2 mg) and 6
(13.7 mg).
1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data:
see Tables 1–3.
3.10. Acid hydrolysis
A solution (1 mg each) of compounds 1, 3 and 5 in 1 N HCl
(0.5 ml) was stirred at 80 °C for 4 h. After cooling, the solution
was concentrated by blowing with N2. The residue was dissolved
in 1-(trimethylsilyl)-imidazole and pyridine (0.1 ml), and the solu-
tion was stirred at 60 °C for 5 min.
3.4. Compound 1
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 analysed by GC using an L-Chirasil-Val column
(0.32 mm ꢂ 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 hydrolysate of 1 were detected
at 10.96 and 12.01 (D-xylose) and 14.72 min (D-glucose). The
peaks of D-glucuronic acid (15.81 min), D-glucose (14.70 min)
and D-xylose (10.94 and 12.00 min) were detected in the hydroly-
sate of 3. The peaks of the hydrolysate of 5 were detected at
14.71 min (D-glucose), 9.67 and 10.70 (L-rhamnose). Retention
times for authentic samples after being treated in the same manner
with 1-(trimethylsilyl)-imidazole in pyridine were detected at
15.81 (D-glucuronic acid), 14.71 min (D-glucose), 9.68 and 10.71
(L-rhamnose), 10.96 and 12.00 min (D-xylose).
Amorphous powder; ½a D25
ꢃ
+34.0 (c 0.1, MeOH); IR (KBr):
m
max = 3480 (>OH), 3025 (cyclopropane ring), 2870 (>CH), 1290–
1030 (C–O–C) cmꢁ1; ESI–MS m/z 809.4 [M+Na]+; MS/MS m/z
629.3 [M+Naꢁ180]+, m/z 479.0 [M+Naꢁ180ꢁ150]+; HRMALDI-
TOFMS [M+Na]+ m/z 809.4669 (calc. for C41H70O14Na, 809.4663);
1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data:
see Tables 1–3.
3.5. Compound 2
Amorphous powder;
½
a 2D5
29.3 (c 0.1, MeOH); IR (KBr):
ꢃ
m
max = 3490 (>OH), 3033 (cyclopropane ring), 2889 (>CH), 1271–
1025 (C–O–C) cmꢁ1; ESI–MS m/z 809.3 [M+Na]+; MS/MS m/z
629.3 [M+Naꢁ180]+; HRMALDITOFMS [M+Na]+ m/z 809.4665 (calc.
for C41H70O14Na, 809.4663); 1H NMR (CD3OD, 600 MHz) and 13C
NMR (CD3OD, 150 MHz) data: see Tables 1–3.
Acknowledgements
We thank Dr. Serdar Sßenol for their valuable assistance for iden-
tification of the plant material. This project was supported by Ege
University Research Foundation (2008 FEN 056).
3.6. Compound 3
Amorphous powder;
½
a 2D5
15.8 (c 0.1, MeOH); IR (KBr):
ꢃ
m
max = 3477 (>OH), 3048 (cyclopropane ring), 2895 (>CH), 1285–
1043 (C–O–C) cmꢁ1; ESI–MS m/z 985.3 [M+Na]+; MS/MS m/z
805.3 [M+Naꢁ180]+, m/z 641 [M+Naꢁ180ꢁ164]+; HRMALDITOFMS
[M+Na]+ m/z 985.4979 (calc. for C47H78O20Na, 985.4984); 1H NMR
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data: see
Tables 1–3.
References
Agrawal, P.K., Jain, D.C., Gupta, R.K., Thakur, R.S., 1985. Carbon-13 NMR
spectroscopy of steroidal sapogenins and steroidal saponins. Phytochemistry
24, 2479–2496.
_
Bedir, E., Çalıßs, I., Aquino, R., Piacente, S., Pizza, C., 1998. Cycloartane triterpene
glycosides from the roots of Astragalus brachypterus and Astragalus
microcephalus. J. Nat. Prod. 61, 1469–1472.
_
3.7. Compound 4
Bedir, E., Çalısß, I., Aquino, R., Piacente, S., Pizza, C., 1999. Trojanoside H:
a
cycloartane-type glycoside from the aerial parts of Astragalus trojanus.
Phytochemistry 51, 1017–1020.
Amorphous powder;
½
a 2D5
22.7 (c 0.1 MeOH); IR (KBr):
ꢃ
_
Bedir, E., Çalıßs, I., Khan, I.A., 2000. Macrophyllosaponin E: a novel compound from
m
max = 3483 (>OH), 3027 (cyclopropane ring), 2877 (>CH), 1279–
the roots of Astragalus oleifolius. Chem. Pharm. Bull. 48, 1081–1083.
1038 (C–O–C) cmꢁ1; ESI–MS m/z 971.4 [M+Na]+; MS/MS m/z
791.4 [M+Naꢁ180]+, m/z 613.3 [M+Naꢁ180ꢁ180]+; HRMALDI-
TOFMS [M+Na]+ m/z 971.5199 (calc. for C47H80O19Na, 971.5192);
1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data:
see Tables 1–3.
_
Bedir, E., Çalısß, I., Dunbar, C., Sharan, R., Buolamwini, J.K., Khan, I.A., 2001a. Two
novel cycloartane-type triterpene glycosides from the roots of Astragalus
prusianus. Tetrahedron 57, 5961–5966.
_
Bedir, E., Tatli, I.I., Çalıßs, I., Khan, I.A., 2001b. Trojanosides I–K: new cycloartane-type
glycosides from the aerial parts of Astragalus trojanus. Chem. Pharm. Bull. 49,
1482–1486.
_
Çalıßs, I., Yuruker, A., Tasdemir, D., Wright, A.D., Sticher, O., Luo, Y.D., Pezzuto, J.M.,
1997. Cycloartane triterpene glycosides from the roots of Astragalus
melanophrurius. Planta Med. 63, 183–186.
3.8. Compound 5
_
Çalıßs, I., Yusufoglu, Z., Zerbe, O., Sticher, O., 1999. Cephalotoside A: a tridesmosidic
cycloartane type glycoside from Astragalus cephalotes var. brevicalyx.
Amorphous powder;
½
a 2D5
17.9 (c 0.1, MeOH); IR (KBr):
ꢃ
Phytochemistry 50, 843–847.
_
m
max = 3472 (>OH), 3040 (cyclopropane ring), 2883 (>CH), 1283–
Çalıßs, I., Dönmez, A.A., Perrone, A., Pizza, C., Piacente, S., 2008. Cycloartane
1029 (C–O–C) cmꢁ1; ESI–MS m/z 985.3 [M+Na]+; MS/MS m/z
805.3 [M+Naꢁ180]+, m/z 641.4 [M+Naꢁ180ꢁ164]+; HRMALDI-
TOFMS [M+Na]+ m/z 985.5350 (calc. for C48H82O19Na, 985.5348);
1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data:
see Tables 1–3.
glycosides from Astragalus campylosema Boiss ssp. campylosema.
Phytochemistry 69, 2634–2638.
Davis, P.H., 1970. Flora of Turkey and East Aegean Islands, vol. 4. University Press,
Edinburgh. pp. 49–254.
Davis, A.M., 1982. Crude protein, crude fiber, tannin, and oxalate concentrations of
33 Astragalus species. J. Range Manage. 35, 32–34.
Fadeev, Y.M., Isaev, M.I., Akimov, Y.A., Kintya, P.K., Gorovits, M.B., Abubakirov, N.K.,
1987. Triterpene glycosides of Astragalus and their genins XXIII.
Cyclocanthogenin from Astragalus tragacantha. Khim. Prir. Soedin. 23, 678–684.
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from the hairy root cultures of Astragalus membranaceus. Phytochemistry 37,
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Isaev, M.I., Gorovits, M.B., Abubakirov, N.K., 1989. Progress in the chemistry of the
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Triterpene glycosides of Tragacantha and their genins. Cyclostipulosides A and B
from Tragacantha stipulosa. Khim. Prir. Soedin. 5, 670–674.
3.9. Compound 6
Amorphous powder;
½
a 2D5
24.4 (c 0.1, MeOH); IR (KBr):
ꢃ
m
max = 3486 (>OH), 3035 (cyclopropane ring), 2874 (>CH), 1292–
1040 (C–O–C) cmꢁ1; ESI–MS m/z 985.4 [M+Na]+; MS/MS m/z
805.3 [M+Naꢁ180]+, m/z 641.3 [M+Naꢁ180ꢁ164]+; HRMALDI-
TOFMS [M+Na]+ m/z 985.5341 (calc. for C48H82O19Na, 985.5348);

634
E. Polat et al. / Phytochemistry 70 (2009) 628–634
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