Saponins from Astragalus hareftae (NAB.) SIRJ.

Article abstract of DOI:10.1016/j.phytochem.2012.07.015

Four cycloartane- (hareftosides A-D) and oleanane-type triterpenoids (hareftoside E) were isolated from Astragalus hareftae along with fifteen known compounds. Structures of the compounds were established as 3,6-di-O-β-d- xylopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (1), 3,6,24-tri-O-β-d-xylopyranosyl-3β,6α,16β,24(S), 25-pentahydroxycycloartane (2), 3-O-β-d-xylopyranosyl-3β,6α, 16β,25-tetrahydroxy-20(R),25(S)-epoxycycloartane (3), 16-O-β-d- glucopyranosyl-3β,6α,16β,25-tetrahydroxy-20(R),24(S) -epoxycycloartane (4), 3-O-[β-d-xylopyranosyl-(1→2)-O-β-d- glucopyranosyl-(1→2)-O-β-d-glucuronopyranosyl]-soyasapogenol B (5) by the extensive use of 1D- and 2D-NMR experiments along with ESI-MS and HR-MS analyses.

Full text of DOI:10.1016/j.phytochem.2012.07.015

Phytochemistry 84 (2012) 147–153
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Saponins from Astragalus hareftae (NAB.) SIRJ.
c
c
d
Ibrahim Horo a, Erdal Bedir b, , Milena Masullo , Sonia Piacente , Fevzi Özgökçe ,
⇑
Özgen Alankußs-Çalıßskan a,
⇑
a
_
Ege University, Faculty of Science, Department of Chemistry, 35100 Bornova, Izmir, Turkey
b
_
Ege University, Faculty of Engineering, Department of Bioengineering, 35100 Bornova, Izmir, Turkey
c Department of Pharmaceutical Sciences, Salerno University, 84084 Fisciano (Salerno), Italy
d Department of Biology, Faculty of Science & Art, Yüzüncü Yıl University, 65080 Van, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2012
Received in revised form 19 July 2012
Available online 25 August 2012
Four cycloartane- (hareftosides A–D) and oleanane-type triterpenoids (hareftoside E) were isolated from
Astragalus hareftae along with fifteen known compounds. Structures of the compounds were established
as 3,6-di-O-b-
pyranosyl-3b,6
hydroxy-20(R),25(S)-epoxycycloartane (3), 16-O-b-
24(S)-epoxycycloartane (4), 3-O-[b- -xylopyranosyl-(1?2)-O-b-
D
-xylopyranosyl-3b,6
,16b,24(S),25-pentahydroxycycloartane (2), 3-O-b-
-glucopyranosyl-3b,6
-glucopyranosyl-(1?2)-O-b-D
a
,16b,24(S),25-pentahydroxycycloartane (1), 3,6,24-tri-O-b-
-xylopyranosyl-3b,6 ,16b,25-tetra-
,16b,25-tetrahydroxy-20(R),
-glucur-
D-xylo-
a
D
a
D
a
Keywords:
Astragalus
Saponin
Cycloartane
Oleanane
Hareftoside
D
D
onopyranosyl]-soyasapogenol B (5) by the extensive use of 1D- and 2D-NMR experiments along with
ESI-MS and HR-MS analyses.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
protozoal (Özipek et al., 2005), wound healing (Sevimli-Gür et al.,
2011) antiviral and cytotoxic activities (Tian et al., 2005).
The Fabaceae (Leguminosae) is a family of flowering plants
comprising 269 genera and 5100 species (Davis, 1970). Astragalus
L., the largest genus in the family Leguminosae, is represented by
445 species in the flora of Turkey and 224 of these species are en-
demic (Davis, 1970; Aytaç, 2000).
Gum tragacanth, a very well known foodstuff and pharmaceuti-
cal emulsifier, is an economically important natural product ob-
tained from Astragalus species growing wild in Turkey (Tang and
Eisenbrand, 1992). In Turkish folk medicine, the aqueous extracts
of some Astragalus species (Sevimli-Gür et al., 2011) are used to
treat leukemia as well as healing wounds (Calis et al., 1997; Bedir
et al., 2000). Roots of these plants are also used as antiperspirant,
diuretic, tonic and for the treatment of nephritis, diabetes, leuke-
mia and uterine cancer (Tang and Eisenbrand, 1992).
Polysaccharides and saponins are the major classes of chemical
compounds isolated from Astragalus species; however, the most
investigated constituents are saponins. Cycloartane- and olean-
ane-type glycosides from Astragalus species show interesting
biological properties, including immune-stimulating (Bedir et al.,
2000; Yesilada et al., 2005; Nalbantsoy et al., 2011, 2012), anti-
Several cycloartane and oleanane-type triterpene glycosides
were isolated from Turkish Astragalus species (Ríos and Waterman,
1997; Verotta and El-Sebakhy, 2001; Bedir et al., 1998a,b, 1999;
Polat et al., 2009, 2010; Horo et al., 2010; Gülcemal et al., 2011).
As part of our studies on the new bioactive compounds from
Turkish Astragalus species, we carried out a study on Astragalus
hareftae (NAB.) SIRJ. (Leguminosae). This paper reports the isola-
tion of four new cycloartane-type (1–4) and a new oleanane-type
triterpene glycosides from the methanolic extract of the whole
plant of A. hareftae along with 11 known cycloartane-type glyco-
sides (6–16) and four known oleanane-type triterpene glycosides
(17–20). Their structures were elucidated by extensive spectro-
scopic methods including 1D- (1H, 13C and TOCSY) and 2D-NMR
(DQF-COSY, HSQC, HMBC, and ROESY) experiments as well as
ESI-MS and HR-MS analyses.
2. Results and discussion
The HR-MALDI-TOF mass spectrum of 1 (m/z 779.4564 [M+Na]+,
calcd. for C40H68O13Na, 779.4558) supported a molecular formula
of C40H68O13. The ESI-MS showed the major ion peak at m/z 779
which was assigned to [M+Na]+. The MS/MS of this ion showed a
peak at m/z 647 [M+Naꢀ132]+, corresponding to the loss of a
⇑
Corresponding authors. Tel./fax: +90 232 3888264 (Ö. Alankusß-Çalıßskan), tel./
pentose unit. In the MS3 spectrum,
a peak at m/z 515
fax: +90 232 3884955 (E. Bedir).
[M+Naꢀ132ꢀ132]+, corresponding to the loss of an additional
E-mail addresses: erdalbedir@gmail.com (E. Bedir), ozgen.alankus.caliska@ege.
edu.tr (Ö. Alankusß-Çalısßkan).
pentose unit, was observed.
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.07.015

148
I. Horo et al. / Phytochemistry 84 (2012) 147–153
In particular, the 1H NMR spectrum of 1 showed characteristic
after the hydrolysis of 1 followed by GC analysis (De Marino et al.,
2003).
On the basis of all the evidence, the structure of compound 1
was established as 3,6-di-O-b- -xylopyranosyl-3b,6 ,16b,24(S),
25-pentahydroxycycloartane, named hareftoside A.
signals due to a cyclopropane methylene as an AX system at dH
0.58 and 0.24 (each 1H, d, J = 4.2 Hz), six tertiary methyl groups
at dH 1.16, 1.17, 1.19, 1.26, 1.02 and 1.00, which were correlated
in HSQC with carbon signals at dC 18.1, 25.2 (x2), 28.0, 16.5 and
19.7, respectively. A secondary methyl group at dH 1.06 (3H, d,
J = 6.5 Hz), and four oxygen bearing methine proton signals at dH
4.48 (ddd, J = 8.0, 8.0, 5.2 Hz), 3.53 (ddd, J = 9.5, 9.5, 4.5 Hz), 3.41
(dd, J = 10.5, 2.2 Hz) and 3.23 (dd, J = 11.3, 4.0 Hz), which were
indicative of secondary alcoholic functions (Table 1), were readily
noticed in the 1H NMR spectrum.
The NMR data of the aglycon moiety of 1 was in good agree-
ment with those reported for cyclocanthogenin (Bedir et al.,
1998a), with glycosidation shifts for C-3 (d 89.8) and C-6 (d 79.3)
(Table 1), thus, confirming the bisdesmosidic structure of 1
(Fig. 1). Additionally, signals of two anomeric protons at dH 4.310
(d, J = 7.5 Hz) and 4.312 (d, J = 7.5 Hz) were observed with 13C
chemical shifts of their relative attached carbons (d 107.5, 105.6,
respectively). The chemical shifts of the rest of the individual pro-
tons of the two sugar units were ascertained from a combination of
1D-TOCSY and DQF-COSY spectral analyses, and 13C chemical shifts
of their relative attached carbons were assigned unambiguously
from the HSQC spectrum (Table 2), substantiating the presence of
two b-xylopyranoside units. Unambiguous determination of the
sequence and linkage sites was obtained from the HMBC spectrum,
which showed key correlation peaks between the proton signal at
dH 4.310 (H-10xyl) and the carbon resonance at dC 89.8 (C-3), and the
proton signal at dH 4.312 (H-100xyl) and the carbon resonance at dC
79.3 (C-6). The D configuration of the xylose units was established
D
a
The HR-MALDI-TOF mass spectrum of 2 (m/z 911.4984 [M+Na]+,
calcd. for C45H76O17Na, 911.4980) supported a molecular formula
of C45H76O17. The ESI-MS spectrum showed the major ion peak at
m/z 911 which was assigned to [M+Na]+. The MS/MS of this ion
showed peaks at m/z 779 [M+Naꢀ132]+, 647 [M+Naꢀ132ꢀ132]+
and 515 [M+Naꢀ132ꢀ132ꢀ132]+, corresponding to the loss of
three pentose units, respectively.
The HSQC, HMBC and COSY spectra of 2 in comparison with
those of 1 showed that 2 differed from 1 by the presence of an
additional xylose unit. The anomeric protons were observed at dH
4.32 (d, J = 7.5 Hz), 4.33 (d, J = 7.5 Hz) and 4.40 (d, J = 7.5 Hz) with
13C chemical shifts of their relative attached carbons at d 107.8,
105.9 and 105.7, respectively. Moreover signal C-24 was found
about +10 ppm downfield shifted, in comparison to that of com-
pound 1. The HMBC correlation between the proton signal at dH
4.33 (H-10xyl) and the carbon resonance at d 89.9 (C-24) allowed
us to determine the linkage site of the additional sugar unit. The
configuration of the three xylose units was established as D after
the hydrolysis of 2 followed by GC analysis (De Marino et al., 2003).
Thus, the structure of 2 was established as 3,6,24-tri-O-b-
xylopyranosyl-3b,6 ,16b,24(S),25-pentahydroxycycloartane, for
which the trivial name hareftoside B is proposed.
D-
a
The molecular formula of 3 was established as C35H58O9 by the
HR-MALDI-TOF mass spectrum (m/z 645.3982 [M+Na]+, calcd. for
Table 1
13C and 1H NMR data of the aglycon moieties of compounds 1–5 (600 MHz, CD3OD).
Position
1
2
3
4
5
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
1
2
3
4
5
6
32.7 1.56, 1.30, m
30.2 1.95, 1.69, m
89.8 3.23, dd (11.3, 4.0)
32.7 1.63, 1.30, m
30.2 1.97, 1.64, m
90.7 3.22, dd (11.3, 4.0)
33.2 1.56, 1.31, m
30.3 1.95, 1.69, m
89.5 3.23, dd (11.3, 4.0)
33.1 1.59, 1.29, m
30.8 1.74, 1.65, m
79.3 3.24, dd (11.3, 4.0)
39.6 1.66, 1.07, m
26.9 2.15, 1.80, m
91.4 3.47, dd (11.5, 4.5)
42.8
–
42.8
–
43.0
–
42.5
–
44.3
–
52.9 1.65, d (9.5)
79.3 3.53, ddd (9.5, 9.5,
4.5)
53.1 1.65, d (9.5)
79.2 3.53, ddd (9.5, 9.5,
4.5)
54.6 1.38, d (9.5)
69.4 3.47, ddd (9.5, 9.5,
4.5)
54.3 1.37, d (9.5)
69.6 3.48, ddd (9.5, 9.5,
4.5)
57.3 0.98, m
19.5 1.67, 1.40, m
7
8
9
10
11
12
13
14
15
39.9 1.72, 1.68, m
45.8 1.91, dd (11.9, 4.2)
39.9 1.71 (2H), m
45.7 1.92, m
38.7 1.46, 1.37, m
48.6 1.83, dd (11.9, 4.2)
38.7 1.51, 1.37, m
48.4 1.88, dd (11.9, 4.2)
34.4 1.58,1.43, m
40.6
48.9 1.61, m
37.4
–
22.1
29.3
–
–
22.0
29.3
–
–
21.7
30.4
–
–
21.8
30.3
–
–
–
26.8 1.84, 1.44, m
33.7 1.79 (2H), m
27.5 1.84, 1.42, m
33.8 1.81 (2H), m
26.9 2.03, 1.21, m
34.5 1.92, 1.73, m
26.9 2.01, 1.25, m
33.6 1.79 (2H), m
47.3
24.8 1.91, (2H) m
123.2 5.28, t (3.5)
46.7
46.8
–
–
46.6
46.8
–
–
47.3
47.3
–
–
145.4
43.3
–
–
47.5
–
47.6 2.08, dd (12.7, 8.0)
1.39, dd (12.7, 5.2)
73.2 4.48, ddd (8.0, 8.0,
5.2)
48.2 2.08, dd (12.7, 8.0)
1.41, dd (12.7, 5.2)
72.8 4.44, ddd (8.0, 8.0,
5.2)
48.3 1.99, dd (12.7, 8.0)
1.49, dd (12.7, 5.2)
74.8 4.64, ddd (8.0, 8.0,
5.2)
48.1 2.10, dd (12.7, 8.0)
1.81, dd (12.7, 5.2)
84.4 4.36, ddd (8.0, 8.0,
5.2)
26.9 1.84, 1.05, m
16
29.8 1.78 (2H), m
17
18
19
57.7 1.73, dd (9.9, 8.0)
18.1 1.16, s
27.7 0.58, d (4.2) 0.24, d
(4.2)
58.1 1.74, dd (9.9, 8.0)
18.0 1.17, s
27.7 0.60, d (4.2) 0.25, d
(4.2)
61.2 2.01, dd (9.9, 8.0)
21.2 1.45, s
32.1 0.57, d (4.2) 0.43, d
(4.2)
60.2 2.35, dd (9.9, 8.0)
21.6 1.45, s
32.1 0.56, d (4.2) 0.39, d
(4.2)
38.6
46.7 2.07, m
47.4 1.77, 0.99, m
–
20
21
22
23
24
29.6 1.91, m
31.2 1.87, m
80.6
28.3 1.55, s
26.9 2.64, 1.21, m
23.8 2.23, 1.72, m
69.7 3.50, brs
–
88.1
26.0 1.44, s
38.5 2.03, 1.93, m
25.8 1.96, 1.88, m
84.6 3.80, t (6.0)
–
37.0
–
18.0 1.06, d (6.5)
33.8 1.81, 1.65, m
28.2 1.64, 1.43, m
78.6 3.41, d (10.3, 2.2)
18.0 0.97, d (6.5)
33.8 1.81, 1.65, m
29.2 1.65 (2H), m
89.9 3.48, dd (10.3, 2.2)
42.1 1.47, 1.36, m
76.8 3.42, m
23.1 1.27, s
64.2 4.17, d (11.5) 3.25,
d (11.5)
25
26
27
28
29
30
73.6
–
73.4
–
76.5
–
72.6
–
16.5 0.94, s
17.6 1.01, s
25.4 1.15, s
20.5 0.87, s
32.6 0.95, s
29.2 1.04, s
25.2 1.17, s
25.2 1.19, s
28.0 1.26, s
16.5 1.02, s
19.7 1.00, s
24.7 1.18, s
26.4 1.18, s
28.1 1.28, s
16.5 1.02, s
19.7 1.00, s
28.6 1.32, s
28.2 1.22, s
28.6 1.33, s
16.4 1.05, s
20.5 0.97, s
25.8 1.21, s
26.7 1.19, s
28.6 1.33, s
15.6 0.98, s
20.6 1.00, s

I. Horo et al. / Phytochemistry 84 (2012) 147–153
149
21
OH
OR
24
25
21
18
18
20
26
20
24
23
25
11
O
17
13
19
OH
OH
19
9
27
16
OH
1
15
10
30
3
7
5
3
O
HO
HO
6
O
O
O
28
29
HO
HO
O
OH
β-D-xyl
OH
O
HO
HO
OH
β-D-xyl
OH
3
β-D-xyl
29
30
1
2
Compound
R
20
12
26
22
18
15
OH
25
O
28
HO
HO
H
9
1
OH
β-D-GlcAO
β-
D
-xyl
27
3
HOOC
HO
HO
7
5
O
O
24
23
HOH2C
HO
HO
HO
O
5
24
20
18
β-D-glc
25
OH
O
O
19
O
HO
16
O
HO
OH
β-D-xyl
3
O
6
OH
HO
β-D-glc
OH
HO
HO
HO
4
Fig. 1. Structures of compounds 1–5.
Table 2
13C and 1H NMR data of the sugar moieties of compounds 1–5 (600 MHz, CD3OD).
1
2
3
4
5
dC
dH (J in Hz)
-Xyl at C-3
dC
dH (J in Hz)
-Xyl at C-3
dC
dH (J in Hz)
-Xyl at C-3
dC
dH (J in Hz)
dC
dH (J in Hz)
b-
D
b-
D
b-
D
b-
D
-Glc (at C-16)
b-
D-GlcA (at C-3)
10
20
30
40
50
107.5 4.31 d (7.5)
75.3 3.21 dd (9.2, 7.5)
77.8 3.32 t (9.2)
71.0 3.51 m
66.5 3.85 dd (11.7, 5.2)
3.20 t (11.7)
107.8 4.32 d (7.5)
75.3 3.21 dd (9.2, 7.5)
77.9 3.33 t (9.2)
71.4 3.51 m
66.8 3.85 dd (11.7, 5.2)
3.20 t (11.7)
106.7 4.30 d (7.5)
75.1 3.21 dd (9.2, 7.5)
77.6 3.31 t (9.2)
71.0 3.49 m
66.4 3.84 dd (11.7, 5.2)
3.20 t (11.7)
106.2 4.28, d (7.5)
104.4 4.51, d (7.5)
75.2 3.20 dd (7.5, 9.0)
78.0 3.36, t (9.0)
71.5 3.32, t (9.0)
77.5 3.26, m
81.1 3.59, dd (7.5, 9.0)
76.8 3.64, dd (9.0, 9.0)
72.7 3.51, dd (9.0, 9.0)
76.6 3.61, d (9.0)
60
62.1 3.86, dd (3.5, 12) 3.71, 176.7
dd (4.5, 12)
–
b-
D
-Xyl at C-6
b-
D
-Xyl at C-6
b-D-Glc (at C-2GlcA)
100
200
300
400
500
105.6 4.312 d (7.5)
75.3 3.21 dd (9.2, 7.5)
77.8 3.32 t (9.2)
71.0 3.51 m
66.5 3.85 dd (11.7, 5.2)
3.20 t (11.7)
105.7 4.40 d (7.5)
75.3 3.21 dd (9.2, 7.5)
77.9 3.33 t (9.2)
71.4 3.51 m
66.8 3.85 dd (11.7, 5.2)
3.20 t (11.7)
103.0 4.84, d (7.5)
83.0 3.75, dd (7.5, 9.0)
74.5 3.68, t (9.0)
70.6 3.86, t (9.0)
76.2 3.52, m
600
62.4 3.82, dd (3.5, 12) 3.75,
dd (4.5, 12)
b-
D
-Xyl at C-24
b-D-Xyl at (at C-2Glc)
1000
2000
3000
4000
5000
105.9 4.33 d (7.5)
75.3 3.21 dd (9.2, 7.5)
77.9 3.33 t (9.2)
71.4 3.51 m
66.8 3.85 dd (11.7, 5.2)
3.20 t (11.7)
106.6 4.58 d (7.5)
75.4 3.31 dd (9.2, 7.5)
76.8 3.41 t (9.2)
70.4 3.55 m
67.0 4.04 dd (11.7, 5.2) 3.29
t (11.7)
C35H58O9Na, 645.3979). The positive ESI-MS spectrum showed the
showed a peak at m/z 513 [M+Naꢀ132]+ due to the loss of a pen-
sodiated ion peak at m/z 645 [M+Na]+. Its MS/MS fragmentation
tose unit.

150
I. Horo et al. / Phytochemistry 84 (2012) 147–153
The 1H NMR spectrum of 3 showed signals due to a cyclopro-
m/z 481 [M+Naꢀ132ꢀ162ꢀ176]+, ascribable to the loss of a glucur-
pane methylene at dH 0.57 and 0.43 (each 1H, d, J = 4.2 Hz), seven
tertiary methyl groups at dH 1.45 (3H, s), 1.55 (3H, s), 1.32 (6H,
s), 1.22 (3H, s), 1.33 (3H, s), 1.05 (3H, s) and 0.97 (3H, s), and four
oxymethine proton signals at dH 4.64 (ddd, J = 8.0, 8.0, 5.2 Hz), 3.47
(ddd, J = 9.5, 9.5, 4.5 Hz), 3.50 (br s) and 3.23 (dd, J = 11.3, 4.0 Hz).
In the 1H NMR spectrum, an anomeric proton at dH 4.30 (d,
J = 7.5 Hz) was observed, implying a monosaccharidic cycloar-
tane-type glycoside. Complete assignments of the 1H and 13C
NMR signals of the sugar portion were accomplished by 1D-TOCSY,
HSQC, HMBC and DQF-COSY experiments, which led to the identi-
fication of a b-xylopyranosyl unit. To clarify the intermolecular
connectivities of the partial structures in 3, HMBC was used. Two
oxygenated quaternary carbons with their characteristic chemical
shifts at d 78.9 and 75.2 (C-20 and C-25, respectively), implying a
monohydroxypyran derivative, were in agreement with those re-
onopyranosyl unit.
In the 1H and 13C NMR spectra of 5, signals derived from seven
methyl groups as singlet at dH 1.27, 1.15, 1.04, 1.01, 0.95, 0.94 and
0.87, an olefinic proton at dH 5.28 (t, J = 3.5 Hz), two oxygen bearing
methine protons at dH 3.47 (dd, J = 11.5, 4.5 Hz), 3.42 (m), and a
primary alcoholic function at dH 3.25 and 4.17 (d, J = 11.5 Hz) were
observed. The NMR spectral data of 5 were consistent with a triter-
pene of the oleanane-type (Kapusta et al., 2005). A detailed inspec-
tion of the 1D and 2D NMR spectra led to the identification of the
aglycon part as soyasapogenol B possessing three sugar residues
(Woldemichael and Winka, 2002). Only the glycosidation shift
was noted for C-3 (d 91.4) (Table 1), confirming the monodesmos-
idic nature of 5. Additionally, in the 1H NMR spectrum, signals
ascribable to three anomeric protons at dH 4.51 (d, J = 7.5 Hz),
4.84 (d, J = 7.5 Hz) and 4.58 (d, J = 7.5 Hz) were noted. The chemical
shifts of the rest of the individual protons of the three sugar units
were ascertained from a combination of 1D-TOCSY and DQF-COSY
spectral analyses, and 13C chemical shifts of their relative attached
carbons were assigned unambiguously from the HSQC spectrum
(Table 2). The data showed the presence of b-glucuronopyranosyl,
b-glucopyranosyl and b-xylopyranosyl units. Determination of the
sugar residue sequence and linkage site was obtained from the
HMBC spectrum, which showed key correlation between the pro-
ton signal at dH 4.51 (H-10GlcA) and the carbon resonance at d
91.4 (C-3), helping to locate b-glucuronopyranosyl unit at the C-3
position of the soyasapogenol B. The C-20 signal of b-glucuronopyr-
anosyl (81.1) was found to be shifted downfield about +5 ppm in
comparison with a terminal b-glucuronopyranosyl moiety (Horo
et al., 2010). In the HMBC spectrum, a cross peak between this car-
ported for 3b,6a,16b,24a-tetrahydroxy-20(R),25-epoxycycloartane
named cyclocephalogenin, previously reported from Astragalus
spp. (Bedir et al., 1998a; Horo et al., 2010; Calis et al., 2001; Alaniya
et al., 2008). The linkage site was deduced based on the key HMBC
correlation between the proton signals at dH 4.30 (H-1’xyl) and the
carbon resonance at d 89.5 (C-3). The hydrolysis of 3 followed by
GC analysis allowed establishing the D configuration of the xylose
unit (De Marino et al., 2003).
Thus, 3 was elucidated as 3-O-b-D-xylopyranosyl-3b,6a,16b,
25-tetrahydroxy-20(R),25(S)-epoxycycloartane, and named as
hareftoside C.
The HR-MALDI-TOF mass spectrum of 4 showed a major ion
peak at m/z 675.4086 [M+Na]+ ascribable to the molecular formula
C
36H60O10 (calcd. for C36H60O10Na, 675.4084). The positive ESI-MS
spectrum showed the sodiated ion peak at m/z 675 [M+Na]+. Its
MS/MS fragmentation showed a peak at m/z 513 [M+Naꢀ162]+
due to the loss of a hexose unit.
bon and H-100
(4.84, d, J = 7.5 Hz) confirmed the inner sugar as
Glc
glucopyranosyl. Also, the C-200glc signal at d 83.0, shifted downfield
about +8 ppm, displayed a cross-peak with the terminal sugar0s
anomeric signal at dH 4.58 (d, J = 7.5 Hz), facilitating b-xylopyrano-
syl unit at C-200. The D configuration of glucose, glucuronic acid and
xylose units was established after the hydrolysis of 5 followed by
GC analysis (De Marino et al., 2003). On the basis of the evidence,
The 1H NMR spectrum of 4 also showed characteristic signals
due to a cycloartane-type triterpenoid framework: cyclopropane
methylene protons at dH 0.56 and 0.39 (each 1H, d, J = 4.2 Hz), se-
ven tertiary methyl groups at dH 1.45 (3H, s), 1.44 (3H, s), 1.33
(3H, s), 1.21 (3H, s), 1.19 (6H, s), 1.00 (3H, s) and 0.98 (3H, s),
and four oxygen bearing methine proton signals at dH 4.36 (ddd,
J = 8.0, 8.0, 5.2 Hz), 3.80 (t, J = 6.0 Hz), 3.48 (ddd, J = 9.5, 9.0,
4.5 Hz) and 3.24 (dd, J = 11.3, 4.0 Hz) (Table 1). The NMR data of
the aglycon of 4 were superimposable with those reported for
cycloastragenol (Kitagawa et al., 1983a), with a glycosidation shift
observed for C-16 (d 84.4) (Table 1), confirming the monodesmos-
idic nature of 4. Additionally, in the 1H NMR spectrum, a signal
ascribable to an anomeric proton at dH 4.28 (d, J = 7.5 Hz) was ob-
served. Chemical shifts of the sugar unit, ascertained from a com-
bination of 1D-TOCSY and DQF-COSY spectral analyses, were
consistent with the presence of a b-glucopyranosyl unit. Determi-
nation of the linkage site was obtained from the HMBC spectrum,
which showed key correlation peak between the proton signal at
dH 4.28 (H-10Glc) and the carbon resonance at d 84.4 (C-16). The D
configuration of the glucose unit was established after the hydro-
lysis of 4 followed by GC analysis (De Marino et al., 2003).
On the basis of these evidence, the structure of compound 4,
the structure of 5 was established as 3-O-[b-D-xylopyranosyl-
(1?2)-O-b- -glucopyranosyl-(1?2)-O-b- -glucuronopyranosyl]-
D
D
soyasapogenol B, for which the trivial name hareftoside E is
proposed.
Additionally, eleven known cycloartane-type glycosides (6–16)
cyclocanthoside E (6) (Bedir et al., 1998b), macrophyllosaponin B
(7) (Calis et al., 1996), 3-O-b-
D-xylopyranosyl-6,25-di-O-b-
D-gluco-
pyranosyl-3b,6 ,16b,24(S),25-pentahydroxycycloartane
a
(8)
(Gülcemal et al., 2011), oleifoloside B (9) (Ríos and Waterman,
1997), cyclocephaloside I (10) (Bedir et al., 1998b), astrasieversia-
nin X (11) (Gan et al., 1986), trojanoside B (12) (Bedir et al., 1999),
cycloastragenol (13) (Kitagawa et al., 1983a), astragaloside IV (14)
(Kitagawa et al., 1983a), brachyoside B (15) (Bedir et al., 1998a),
cyclodissectoside (16) (Sukhina et al., 2007), and four known ole-
anane-type triterpene glycosides, azukisaponin V (17) (Kitagawa
et al., 1983a), dehydroazukisaponin V (18) (Mohamed et al.,
1995), wistariasaponin D (19) (Konoshima et al., 1991), astragalo-
side VIII (20) (Kitagawa et al., 1983b), were isolated.
which was named as hareftoside D, was established as 16-O-b-
glucopyranosyl-3b,6a,16b,25-tetrahydroxy-20(R),24(S)-epoxycycl-
oartane.
D
-
Consequently, as part of our ongoing project on the Astragalus
genus, we investigated the glycosidic constituents of A. hareftae.
We have shown that A. hareftae, which belongs to Acanthophace
section, contains cycloartane and oleanane type triterpenoids. Un-
til now, 25 out of 445 Turkish Astragalus species, from 13 different
sections, have been investigated for their secondary metabolite
contents. Cycloastragenol, possessing 20(R),24(S)-epoxy side chain,
is the chief aglycone in the Astragalus genera. However, cyclocep-
halogenol is more unusual in the genus so far reported only from
3 sections, viz., Rhacophorus, Adiaspastus and Acanthophace. From
The HR-MALDI-TOF mass spectrum of 5 showed a major ion
peak at m/z 951.4932 [M+Na]+ ascribable to the molecular formula
C47H76O18 (calcd. for C47H76O18Na, 951.4929). The ESI-MS spec-
trum showed the major ion peak at m/z 951, which was assigned
to [M+Na]+. The MS/MS of this ion showed peaks at m/z 819
[M+Naꢀ132]+, corresponding to the loss of a pentose unit, at m/z
657 [M+Naꢀ132ꢀ162]+ due to the loss of a hexose unit and at

I. Horo et al. / Phytochemistry 84 (2012) 147–153
151
the Rhacoporus section, which is the largest section of Astragalus
with about 100 members, three out of eight studied Turkish spe-
cies gave cyclocephalogenol (A. microcephalus, Bedir et al., 1998b;
A. zahlbruckneri, Calis et al., 2001; A. schottianus, Karabey et al.,
2012), whereas A. aureus, single examined species of Adiaspastus
section, provided the same aglycone. Only two members of
Acanthophace section, viz., A. hareftae and A. icmadophilus, have
been studied so far, and it is noteworthy that cyclocephalogenol
is encountered in both members. This suggests that the presence
of cyclocephalogenol in Acanthophace section could be of taxo-
nomic importance. Further studies are required to confirm the
above assumption, and our continuing studies will be of use in
clarifying chemotaxonomical classification of the genus Astragalus.
1000 ml; 2:8, 800 ml) and MeOH (1000 ml) to afford six main frac-
tions (Fr.1A–Fr.1F).
Fr.1E (30 mg) was applied to si gel (Merck, 7734, 20 g) column
chromatography. Elution was carried out under isocratic condition
with CHCl3–MeOH–H2O (90:10:1) to give 11 (4.6 mg) and 14
(15 mg). Fr.1D (1.695 g) was subjected to si gel (65 g) open column
chromatography, employed with CHCl3–MeOH (95:5 and 90:10)
and CHCl3–MeOH–H2O mixtures (90:10:1 and 80:20:2) to afford
six sub-fractions (Fr.1Da–Fr.1Df).
Fr.1Dc (49 mg) was chromatographed on RP (C-18) (22 g) by
using MeOH–H2O (6:4) to yield 2 (7.4 mg) and 12 (6 mg). Fr.1De
(28 mg) was subjected to open column chromatography using RP
(C-18) (28 g), eluted with MeOH–H2O (4:6) to give 5 (4 mg), 18
(5 mg) and 19 (5 mg). Fr.1Df (150 mg) was applied to reverse-
phase material (C-18, 28 g), eluted with H2O–MeOH (5:5) to afford
17 (6.5 mg) and 20 (6.5 mg).
3. Experimental
The other fractions to be studied were lost due to a laboratory
accident. Therefore, the remaining part of the plant material
(1.2 kg) was studied by using the same methodology (n-BuOH ex-
tract: 25 g) to obtain the other saponins. The BuOH extract was
subjected to VLC using reverse-phase material (300 g) eluted with
water (1200 ml), H2O–MeOH (7:3, 1800 ml; 5:5, 4000 ml; 3:7,
3000 ml and MeOH 1000 ml) to give six main fractions (Fr.G–Fr.L).
Fr.L (1.5 g) was chromatographed over si gel (240 g), eluted
with CH2Cl2–MeOH–H2O (90:10:1) yielding six sub-fractions.
Fr.L1 (48 mg) was subjected to si gel (17 g) column chromatogra-
phy. Elution was carried out with CH2Cl2–MeOH (95:5) to obtain
13 (12 mg). Fr.L3 (103 mg) was chromatographed on the medium
pressure liquid chromatography (MPLC) by using RP-18 with the
solvent system H2O–MeOH (4:6) to afford 15 (14.3 mg). Fr.L4
(48 mg) was subjected to si gel (32 g) column chromatography,
eluted with CH2Cl2–MeOH–H2O (90:10:1) to give 6 (10.4 mg).
Fr.J (4.8 g) was subjected to MPLC. Elution was started with the
solvent system H2O–MeOH (5:5), increased step wise to H2O–
MeOH (2:8) to yield six subfractions (Fr.J1–Fr.J6). Fr.J4 (170 mg)
was subjected to si gel (45 g) column chromatography, employed
with CH2Cl2–MeOH–H2O (90:10:1) to give 1 (16 mg) and a sub-
fraction Fr.J4.1 (30 mg). Fr.J4.1 was applied to si gel (17 g) once
more with solvent system CH2Cl2–MeOH–H2O (80:20:2) to afford
9 (21 mg). Fr.J3 (850 mg) was subjected to si gel (50 g) column
chromatography with the solvent system CH2Cl2–MeOH–H2O
(90:10:1) to give 3 (3 mg), 4 (3.1 mg), 16 (4.4 mg), 8 (20.5 mg),
and a sub-fraction Fr.J3.1 (190 mg). Fr.J3.1 was subjected to MPLC
(RP, C-18), employed with solvent system H2O–MeOH (5:5) to af-
ford 10 (94 mg) and 7 (21.4 mg).
3.1. General
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 300K. All 2D-NMR
spectra were acquired in CD3OD (99.95%, Sigma–Aldrich) and stan-
dard 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 the matrix-assisted
laser desorption ionization time-of-flight (MALDI-TOF) 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 ꢁ 25 m).
3.2. Plant material
A. hareftae (NAB.) SIRJ. was collected from the altitude of 3200 m
in Baßset Mountain in Gürpınar Village, Van, Eastern Anatolia, Turkey
on July 25, 2008. The plant material was identified by F. Özgökçe
(Department of Biology, Faculty of Science & Art, Yüzüncü Yıl Uni-
versity, Van, Turkey). Voucher specimen has been deposited in the
Herbarium of Yüzüncü Yıl University, Van, Turkey (VANF 13189).
3.4. 3,6-Di-O-b-
D-xylopyranosyl-3b,6a,16b,24(S),25-
pentahydroxycycloartane (1)
Amorphous white solid; ½a D25
ꢂ
+27.3 (c 0.1 MeOH); IR
m
cmꢀ1
:
KBr
max
3.3. Extraction and isolation
3460 (>OH), 3035 (cyclopropane ring), 2945 (>CH), 1260 and 1058
(C–O–C); 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD,
150 MHz) data of aglycone moiety, see Table 1; 1H NMR (CD3OD,
600 MHz) and 13C NMR (CD3OD, 150 MHz) data of the sugar por-
tion, see Table 2; ESI-MS m/z 779 [M+Na]+; MS/MS m/z 647
[M+Naꢀ132]+, m/z 515 [M+Naꢀ132ꢀ132]+; HR-MALDI-TOF-MS
m/z [M+Na]+ calcd. for C40H68O13Na, 779.4558, found 779.4564.
Part of the air-dried and powdered plant material of A. hareftae
(whole plant; 1.6 kg out of 2.8 kg) was first extracted with n-hex-
ane (1 ꢁ 4 L) under reflux at 68 °C, and then with MeOH (3 ꢁ 4 L)
under reflux at 64 °C for 6 h (each). After filtration and evaporation
procedures, n-hexane (3.85 g) and MeOH (50 g) extracts were ob-
tained respectively. The MeOH extract (50 g) was dissolved in
water (300 ml) and partitioned with CH2Cl2 (3 ꢁ 200 ml) and n-
BuOH (3 ꢁ 200 ml), respectively. The n-BuOH phase (5.45 g), which
was rich in saponins, was subjected to open column chromatogra-
phy by using Sephadex LH-20 (150 g), eluted with MeOH to give
seven main fractions (Fr.1–Fr.7). Fr.1 (4.05 g) was subjected to vac-
uum liquid chromatography (VLC), using reverse-phase material
3.5. 3,6,24-Tri-O-b-
D-xylopyranosyl-3b,6a,16b,24(S),25-
pentahydroxycycloartane (2)
Amorphous white solid; ½a D25
ꢂ
+24.3 (c 0.1 MeOH); IR
m
cmꢀ1
:
max
KBr
3470 (>OH), 3030 (cyclopropane ring), 2950 (>CH), 1264 and 1060
(C–O–C); 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD,
150 MHz) data of aglycone moiety, see Table 1; 1H NMR (CD3OD,
600 MHz) and 13C NMR (CD3OD, 150 MHz) data of the sugar
(Lichroprep RP-18, 25–40
lm, 130 g). Elution was performed with
water (600 ml), H2O–MeOH (8:2, 900 ml; 6:4, 1300 ml; 4:6,

152
I. Horo et al. / Phytochemistry 84 (2012) 147–153
portion, see Table 2; ESI-MS m/z 911 [M+Na]+; MS/MS m/z 779
Appendix A. Supplementary data
[M+Naꢀ132]+,
m/z
647
[M+Naꢀ132ꢀ132]+,
m/z
515
[M+Naꢀ132ꢀ132ꢀ132]+; HR-MALDI-TOF-MS m/z [M+Na]+ calcd.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2012.
07.015.
for C45H76O17Na, 911.4980, found 911.4984.
3.6. 3-O-b-D-xylopyranosyl-3,6,16,25-tetrahydroxy-20(R),25(S)-
epoxycycloartane (3)
References
Amorphous white solid; ½a D25
ꢂ
+19.8 (c 0.1 MeOH); IR
m
cmꢀ1
:
max
KBr
Alaniya, M.D., Kavtaradze, N.Sh., Faure, R., Debrauwer, L., 2008. Cycloascauloside B
from Astragalus caucasicus. Chem. Nat. Compd. 44, 324–326.
Aytaç, Z., 2000. Astragalus L. In: Güner, A., Ozhatay, N., Ekim, T., Baßsar, K.H.C. (Eds.),
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Bedir, E., Calis, I., Aquino, R., Piacente, S., Pizza, C., 1998a. Cycloartane triterpene
glycosides from the roots of Astragalus brachypterus and Astragalus
microcephalus. J. Nat. Prod. 61, 1469–1472.
3480 (>OH), 3030 (cyclopropane ring), 2945 (>CH), 1260 and 1055
(C–O–C); 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD,
150 MHz) data of aglycone moiety, see Table 1; 1H NMR (CD3OD,
600 MHz) and 13C NMR (CD3OD, 150 MHz) data of the sugar por-
tion, see Table 2; ESI-MS m/z 645 [M+Na]+; MS/MS m/z 513
[M+Naꢀ132]+; HR-MALDI-TOF-MS m/z [M+Na]+ calcd. for
Bedir, E., Calis, I., Zerbe, O., Sticher, O., 1998b. Cyclocephaloside I:
a novel
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503–505.
C
35H58O9Na, 645.3979, found 645.3982.
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a
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Bedir, E., Pugh, N., Calis, I., Pasco, D.S., Khan, I.A., 2000. Immunostimulatory effects
of cycloartane-type triterpene glycosides from Astragalus species. Biol. Pharm.
Bull. 23, 834–837.
3.7. 16-O-b-
epoxycycloartane (4)
D-glucopyranosyl-3,6,16,25-tetrahydroxy-20(R),24(S)-
Amorphous white solid; ½a D25
ꢂ
+20.8 (c 0.1 MeOH); 3460 (>OH),
Calis, I., Zor, M., Saracoglu, I., Isimer, A., Ruegger, H., 1996. Four novel cycloartane
glycosides from Astragalus oleifolius. J. Nat. Prod. 59, 1019–1023.
Calis, I., Yürüker, A., Tasßdemir, D., Wright, A.D., Sticher, O., Luo, Y.D., Pezzuto, J.M.,
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3040 (cyclopropane ring), 2935 (>CH), 1250 and 1050 (C–O–C);
1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data
of aglycone moiety, see Table 1; 1H NMR (CD3OD, 600 MHz) and
13C NMR (CD3OD, 150 MHz) data of the sugar portion, see Table 2;
ESI-MS m/z 675 [M+Na]+; MS/MS m/z 513 [M+Naꢀ162]+; HR-MAL-
DI-TOF-MS m/z [M+Na]+ calcd. for C36H60O10Na, 675.4084, found
675.4086.
_
Calis, I., Gazar, H.A., Piacente, S., Pizza, C., 2001. Secondary metabolites from the
roots of Astragalus zahlbruckneri. J. Nat. Prod. 64, 1179–1182.
Davis, P.H., 1970. Flora of Turkey and East Aegean Islands, vol. 3. Edinburgh
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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
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3.8. 3-O-[b-
D-xylopyranosyl-(1?2)-O-b-D-glucopyranosyl-(1?2)-O-
Gan, L., Han, X., Chen, Y., 1986. The chemical investigation of Astragalus sieversianus.
Phytochemistry 25, 1437–1441.
b-D-glucuronopyranosyl] soyasapogenol B (5)
Gülcemal, D., Alankus-Caliskan, Ö., Perrone, A., Özgökce, F., Piacente, S., Bedir, E.,
2011. Cycloartane glycosides from Astragalus aureus. Phytochemistry 72, 761–
768.
Horo, I., Bedir, E., Perrone, A., Özgökce, F., Piacente, S., Alankus-Caliskan, Ö., 2010.
Triterpene glycosides from Astragalus icmadophilus. Phytochemistry 71, 956–
973.
Kapusta, I., Stochmal, A., Perrone, A., Piacente, S., Pizza, C., Oleszek, W., 2005.
Triterpene saponins from barrel medic (Medicago truncatula) aerial parts. J.
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Karabey, F., Khan, I.A., Bedir, E., 2012. Cycloartane-type glycosides from Astragalus
schottianus. Phytochem. Lett. 5, 320–324.
Amorphous white solid; C53H84O24; ½a D25
ꢂ
+12.1 (c 0.1 MeOH); IR
cmꢀ1: 3448 (>OH), 2934 (>CH), 1658 (C@C); 1H NMR (CD3OD,
KBr
max
m
600 MHz) and 13C NMR (CD3OD, 150 MHz) data of aglycone moi-
ety, see Table 1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD,
150 MHz) data of the sugar portion, see Table 2; ESI-MS m/z 951
[M+Na]+;
MS/MS
m/z
819
[M+Naꢀ132]+,
m/z
657
[M+Naꢀ132ꢀ162]+, 481 [M+Naꢀ132ꢀ162ꢀ176]+; HR-MALDI-
TOF-MS m/z [M+Na]+ calcd. for C47H76O18Na, 951.4929, found
951.4932.
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3.9. Acid hydrolysis
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54, 830–836.
The configurations of sugar units were established after the
hydrolysis of 1–5 with 1 N HCl, trimethylsilation and determina-
tion of the retention times by GC operating in the experimental
conditions previously reported by De Marino et al. (2003).
The peaks of the hydrolysate of 1–3 were detected at 10.99 and
Mohamed, K.M., Ohtani, K., Kasai, R., Yamasaki, K., 1995. Oleanene glycosides from
seeds of Trifolium alexandrinum. Phytochemistry 40, 1237–1242.
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Nalbantsoy, A., Nesil, T., Erden, S., Calis, I., Bedir, E., 2011. Adjuvant effects of
Astragalus saponins macrophyllosaponin
B
and astragaloside VII. J.
12.02 (
glucose) was detected. The peaks of the hydrolysate of 5 were de-
tected at 15.82 min ( -glucuronic acid), 14.73 min ( -glucose), and
10.98 and 12.02 ( -xylose). Retention times for authentic samples
after being treated in the same manner with 1-(trimethylsilyl)-
imidazole in pyridine were detected at 10.98 and 12.00 min ( -xy-
-glucuronic
D-xylose). For the hydrolysate of 4 a peak at 14.73 min (D-
Ethnopharmacol. 134, 897–903.
Nalbantsoy, A., Nesil, T., Dilsiz, O.Y., Aksu, G., Khan, S., Bedir, E., 2012. Evaluation of
the immunomodulatory properties in mice and in vitro anti-inflammatory
activity of cycloartane type saponins from Astragalus species. J.
Ethnopharmacol. 139, 574–581.
Özipek, M., Dönmez, A.A., Calis, I., Brun, R., Rüedi, P., Tasdemir, D., 2005.
Leishmanicidal cycloartane-type triterpene glycosides from Astragalus
oleifolius. Phytochemistry 66, 1168–1173.
D
D
D
D
lose), at 14.71 min (
acid).
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