Cycloartane-type glycosides from Astragalus schottianus

Article abstract of DOI:10.1016/j.phytol.2012.02.011

Three new cycloartane-type triterpene glycosides were isolated from the roots of Astragalus schottianus Boiss. Their structures were established as 20(R),25-epoxy-3-O-β-d-xylopyranosyl-24-O-β-d-glucopyranosyl-3β, 6α,16β,24α-tetrahydroxycycloartane (1), 20

Full text of DOI:10.1016/j.phytol.2012.02.011

Phytochemistry Letters 5 (2012) 320–324
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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Cycloartane-type glycosides from Astragalus schottianus
Fatih Karabey a, Ikhlas A. Khan b,c, Erdal Bedir a,
*
a
˙
Department of Bioengineering, Faculty of Engineering, Ege University, 35100 Bornova-Izmir, Turkey
b National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
c Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
A R T I C L E I N F O
A B S T R A C T
Article history:
Three new cycloartane-type triterpene glycosides were isolated from the roots of Astragalus schottianus
Received 11 November 2011
Received in revised form 20 February 2012
Accepted 25 February 2012
Available online 22 March 2012
Boiss. Their structures were established as 20(R),25-epoxy-3-O-
anosyl-3 ,6 ,16 ,24 -tetrahydroxycycloartane (1), 20(R),25-epoxy-3-O-[
-xylopyranosyl-24-O- -glucopyranosyl-3 ,6 ,16 ,24 -tetrahydroxycycloartane (2), 3-O-
xylopyranosyl-3 ,6 ,16 ,20(S),24(S),25-hexahydroxycycloartane (3) by the extensive use of 1D and
2D-NMR techniques and mass spectrometry.
ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
b
-
D
-xylopyranosyl-24-O-
b-D-glucopyr-
b
a
b
a
b-D-glucopyranosyl(1 ! 2)]-
b
-D
b
b
-
D
b
a
b
a
b-D-
b
a
Keywords:
Astragalus schottianus
Saponin
Cycloartane glycoside
Structure elucidation
Cyclocephalogenol
Schottianogenol
1. Introduction
describes the isolation and structure elucidation of three new
cycloartane-type glycosides.
Among Turkish flora, the Astragalus genus is represented by 445
species, of which 224 are endemic to Turkey (Davis, 1982; Aytac¸,
2000). Polysaccharides and saponins are the major classes of
chemical compounds that have been isolated from Astragalus
species. The polysaccharides are reported to possess cytotoxic and
immunostimulating effects (Bedir et al., 2000b; Rios and Water-
man, 1997), but the most thoroughly investigated constituents of
Astragalus are the saponins.
2. Results and discussion
High-resolution electrospray ionization mass spectrometry
(HRESIMS) of 1 showed a base peak at m/z 807.46042 [M+Na]+.
This result supported a molecular formula of C41H68O14 (m/z
807.45068).
The 1H NMR spectrum of 1 showed characteristic signals due to
In the course of studies on Turkish Astragalus species, several
cycloartane- and oleanane-type triterpene glycosides were isolat-
ed and their structures elucidated (Calis et al., 1997, 1999; Bedir
et al., 1999; Polat et al., 2009, 2010; Horo et al., 2010; Gu¨lcemal
et al., 2011).
Cycloartane- and oleanane-type glycosides from Astragalus
species have shown interesting biological properties, including
immunostimulating (Bedir et al., 2000b; Calis et al., 1997; Yesilada
cyclopropane–methylene protons as an AX system (d 0.12 and
0.49, JAX = 4.0 Hz; H2-19) and seven tertiary methyl groups.
Additionally, the resonances for two anomeric protons were
observed at
d 4.90 (d, J = 7.2 Hz) and 4.88 (d, J = 7.6 Hz). Thus,
compound 1 was considered to be a cycloartane-type triterpene
diglycoside (Fig. 1). The NMR signals were analyzed by the use of
COSY and HMQC. The 1H and 13C NMR data supported the
assignment of the sugar moieties in 1 as
b-xylopyranose and b-
¨
et al., 2005), anti-protozoal (Ozipek et al., 2005), antiviral
glucopyranose. The remaining carbon and proton resonances were
consistent with C30H50O5 for the aglycon moiety. This result
indicated six saturated ring systems because there were no olefinic
protons. Additional functionalities on the aglycon included four
geminal methine protons on the oxygen-bearing carbon atoms (H-
3, H-6, H-16, and H-24). The resonances for the oxygenated
(Gariboldi et al., 1995), wound healing (Sevimli-Gur et al.,
2011), adjuvant (Nalbantsoy et al., 2011) and cytotoxic activities
(Radwan et al., 2004).
In our continuing studies of the constituents of Astragalus
species, we investigated the roots of A. schottianus. This paper
carbons also indicated the presence of four oxymethine carbons (
89.2, 68.7, 74.8, 74.7; C-3, C-6, C-16, and C-24, respectively) and
two oxygenated quaternary carbons ( 79.9 and 75.5, C-20 and C-
25, respectively). HMBC was used to clarify the intermolecular
d
d
* Corresponding author. Tel.: +90 232 388 4955; fax: +90 232 388 4955.
E-mail address: erdal.bedir@ege.edu.tr (E. Bedir).
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2012.02.011

F. Karabey et al. / Phytochemistry Letters 5 (2012) 320–324
321
Fig. 1. Structures of 1–3.
connectivity of the partial structures in 1. This experiment revealed
not only connectivity but also interglycosidic linkages. These data
suggested the presence of a monohydroxypyran ring as a side
chain. This configuration is unusual in the plant kingdom, so far it
has been reported only from Astragalus spp. Based on these data, it
was deduced that compound 1 was a cycloartane diglycoside with
cyclocephalogenol as the aglycon previously reported by our group
(Bedir et al., 1998). Additionally, the results of the HMBC
experiment suggested that compound 1 was a bisdesmosidic
saponin in which the sugar residues were linked to C-3 and C-24 of
cyclocephalogenol. Key correlation peaks observed in the HMBC
case of glycosylation at position 6, the carbon resonance was
observed around 79.0 ppm that is supposed to provide a long-
range correlation with the anomeric proton in the HMBC spectrum.
Interestingly, the authors state that the HMBC correlation between
78.7 ppm (assigned to C-24) and 4.86 ppm (anomeric proton of b-
glucose) signals facilitates the sugar residue being attached to C-
24(O) position. But in Semmar’s HMBC table providing evidence for
the proposed structure, key interactions which should be observed
between C-24 (or H-24) and CH3-26, CH3-27, CH2-23, CH2-22, and
from C-6 (or H-6) to CH-5, CH2-7 and CH2-8 were not mentioned,
whereas we observed and reported these very essential long range
correlations for compound 1. The data indicated that the C-6 (H-6)
and C-24 (H-24) resonances were interpreted incorrectly by
Semmar and his coworkers implying a known structure, cycloce-
phaloside I, reported by our group in 1998 (Bedir et al., 1998).
The molecular formula of 2, was established as C47H78O20 by
HRESIMS analysis (m/z 981.48275 [M-Cl]-, calcd for C47H78O20Cl,
981.48258).
spectrum of 1 between H-10 of the xylosyl at
and C-3 (
89.2) of the aglycon and between H-100 of the glucosyl at
4.88 (d, J = 7.6 Hz) and C-24 ( 74.7) allowed the saccharide
d 4.90 (d, J = 7.2 Hz)
d
d
d
residues at C-3 and C-24. The D configuration of the xylose and
glucose units was determined after acidic hydrolysis of 1 followed
by gas chromatography analysis. Consequently, the structure of 1
was established as 20(R),25-epoxy-3-O-
-glucopyranosyl-3 ,6 ,16 ,24 -tetrahydroxycycloartane.
The structure of 1 was previously reported by Semmar et al.
b-D-xylopyranosyl-24-O-
b
-D
b
a
b
a
The 1H NMR spectrum of 2 showed three anomeric proton
resonances at
J = 7.8 Hz) correlated by HSQC to the resonances at
and 100.7, respectively. The three sugar units were identified
using a combination of COSY and HSQC as two terminal
glucopyranoses, and glycosylated -xylopyranose. The D-con-
d
4.90 (d, J = 8.0 Hz), 4.92 (d, J = 7.2 Hz), and 5.40 (d,
(2001) as a new natural product. However, on the basis of our
detailed inspections, we concluded that the researchers may have
misinterpreted their NMR data and proposed an incorrect
structure. The correct structure should have been determined as
cyclocephaloside I (Bedir et al., 1998). Our conclusion is supported
by the following facts: In the case of glycosylation at C-6 position,
C-7 gives a resonance at about 33.0–34.0 ppm as a result of
d
106.0, 105.6
b
-
b
figurations of the sugar residues were determined by acid
hydrolysis. The 13C NMR resonances arising from the sapogenol
moiety were identical to those of 1, exhibiting same glycosyla-
glycosylation’s up field shift on
b
-carbon. If there is no such
tion pattern on the aglycone. These results suggested a
linkage, C-7 is observed around 38.5–39.0 ppm (Agzamova and
Isaev, 1999; Bedir et al., 1998). In Semmar’s paper; the carbon
signal at 32.0 ppm was assigned to C-7, whereas our NMR spectra
of 1 indicate C-7 at 39.3 ppm. Same pattern should also be
considered for C-23 resonance due to glycosylation at C-24
bidesmosidic structure for 2 in which the two sugar units were
attached to the hydroxyl groups at C-3 and C-24. HMBC
experiment performed on 2 established the glycosylation sites
showing significant cross-peaks, due to 2JC–H correlations,
0
0
(
between C-3 (
d
88.6) and XylH-1
(
d
3.55), between XylC-2
d
00
position (
d
C-23 = 26.8 for Semmar;
d
C-23 = 21.8 for 1). Additionally,
70.4 and 78.7. In the
83.3) and Glu-IH-1
(
d
4.90), and between C-24 (
d
74.6) and Glu-
000
C-6 and C-24 resonances were reported at
d
IIH-1
(
d
5.40). Consequently, the structure of 2 was established

322
F. Karabey et al. / Phytochemistry Letters 5 (2012) 320–324
as
20(R),25-epoxy-3-O-[
b
-
D
-glucopyranosyl(1!2)]-
b
-
D
-xylo-
configurations of C-20 and C-24. 13C NMR data of 3 are comparable
to those reported for analogous compounds having a 20(S), 24(R)
configuration (Duc et al., 1994; Fadeev et al., 1987; Rong-Qi et al.,
1991; Sun and Chen, 1997). In the case of 20(S) configuration dC-21
gives resonance at 27.0–28.0 ppm (Duc et al., 1994; Rong-Qi et al.,
1991; Sun and Chen, 1997; Yalc¸ın et al., 2012), while 20(R)
configuration gives dC-21 at 22.0–22.5 ppm (Duc et al., 1994) (dC-
21 = 28.7 for 3). In the case of 24(R) configuration dC-24 gives
resonance around 80.0 ppm (Duc et al., 1994; Fadeev et al., 1987),
whereas for the 24(S) configuration dC-24 resonates at 77.0–
pyranosyl-24-O-
droxycycloartane, a new natural product.
b
-
D
-glucopyranosyl-3
b
,6 ,16 ,24 -tetrahy-
a
b
a
HRESIMS [ESI(+)MicroTOF] of 3 (m/z 675.38677) [M-Cl]ꢀ, calcd
for C35H60O10Cl, 675.38750) provided a molecular formula of
C
35H60O10
.
Taking into account the results of our 1H and 13C NMR studies,
the main features of a cyclopropane-type triterpene possessing an
acyclic side chain were evident for compound 3: characteristic
signals due to cyclopropane–methylene protons as an AX system (
d
0.28, 0.58, JAX = 4.0 Hz, H2-19), and seven tertiary methyl groups (
d
77.2 ppm (Bedir et al., 2000a; Duc et al., 1994) (dC-24 = 79.9 for 3).
2.02, 1.79, 1.03, 1.35, 1.57, 1.54, 1.68; respectively, H3-29, H3-18,
H3-30, H3-28, H3-27, H3-26, H3-21). Inspection of the 1H NMR
spectral data of 3 showed seven tertiary methyl groups (d 2.02,
Additionally, in the ROESY spectrum, a correlation between H-16
and H3-21 demonstrated their co-facial nature (alpha orientation),
and verified 20(S) absolute configuration.
1.79, 1.03, 1.35, 1.57, 1.54, 1.68; respectively, H3-29, H3-18, H3-30,
H3-28, H3-27, H3-26, H3-21). Additionally, the resonance for an
Based on these evidence, the structure of 3 was established as 3-
O-b-D-xylopyranosyl-3b,6a,16b,20(S),24(R),25-hexahydroxycy-
anomeric proton was observed at
d
4.61 (J = 7.2 Hz) (H-1xyl),
cloartane (Fig. 1). This compound represents the second entry of
the series of cycloartane-type compound possessing a 20-OH
functional group in Astragalus genus. In nature, only five
compounds, obtained from Oxytropis bicolor and Astragalus
stereocalyx, were reported to have such a substitution at C-20
indicative of the presence of a -linked sugar unit. Thus, compound
b
3 was considered to be a cycloartane-type triterpene monoglyco-
side.
The 13C NMR spectrum of 3 contained 35 signals. Five signals
were assigned to the sugar unit, which were in good accordance
and 3b,16b,20(S),24(S),25-pentahydroxycycloartane framework
with the presence of
b
-xylopyranose. On acidic hydrolysis of 3, D
(Rong-Qi et al., 1991; Sun and Chen, 1997; Yalc¸ın et al., 2012).
configuration of the xylose unit was confirmed. The remaining 30
resonances were consistent with a C30H52O6 triterpene framework,
indicating the presence of 5 degrees of unsaturation. This implied
five saturated ring systems because there were no olefinic protons
(A–D rings and a cyclopropane ring). Thus, compound 3 was readily
deduced to be a cycloartane possessing an acyclic side chain. 13C-
and DEPT spectra showed that the resonances assigned to the
aglycon moiety consist of 7 methyl, 9 methylene, 7 methine; four
3. Experimental
3.1. General
HR-MS spectra were obtained by microToF-Q II mass spec-
trometer from Bruker (Bremen, Germany), in positive and negative
ESI mode. The 1D and 2D NMR spectra were obtained on Varian
Oxford AS400 at 400 (1H) and 100 MHz (13C) instruments. Proton
and carbon chemical shifts were reported relative to TMS. 2D NMR
spectra (COSY, ROESY, HMQC, HMBC) were run using standard
Varian pulse programs. GC analysis was performed on a Termo
Finnigan Trace GC apparatus using an L-Chirasil-Val column
(0.32 mm ꢁ 25 m). Column chromatography was carried out on
of which were oxygen-bearing (
quaternary carbons; two of which were oxygen-bearing (
d
67.7, 75.5, 79.6 and 88.4), and 7
72.6
d
and 76.7). Full assignment of the 1H and 13C signals of the aglycon
part of 3 accomplished by COSY and HMQC spectra. From the DQF-
COSY and HMQC experiments, it was possible to establish 5
sequences excluding the signals ascribable to the cyclopropane
ring (H2-19):–CH2–CH2–CH(O),–CH–CH(O)–CH2–CH–,–CH2–CH2–
,–CH2–CH(O)–CH–,–CH2–CH2–CH(O)–, corresponding to the C-
1 ! C-3, C-5 ! C-8, C-11 ! C-12, C-15 ! C-17 and C-22 ! C-24
moieties, respectively (Fig. 2). All connectivity within 3 was also
confirmed by HMBC spectrum (Fig. 2), which showed a long-range
silica gel (JT Baker, 40
ciences, 17-0090-02) and RP (C-18, 40
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 105 8C for 1–2 min.
m
m), Sephadex LH-20 (Amersham Bios-
m) (Merck). TLC analyses
m
correlation between the anomeric proton signal at
d 4.61 (d,
J = 7.2 Hz) and the carbon resonance at 88.4 (C-3), confirming the
d
3.2. Plant material
location of glycosidation. Additionally, the oxygenated quaternary
carbons were located at C-20 and C-25 based on the major cross
peaks between C-20 ! H3-21 and C-25 ! H3-26/H3-27 (Fig. 2).
Analyses of H–H vicinal coupling constants and NOE interac-
Astragalus schottianus Boiss. was collected from Zara Village,
10 km from Erzincan-Sivas highway, Central Anatolia, Turkey in
July 20, 2008. Voucher specimen has been deposited in the
Herbarium of Bionorm Natural Products Company, Izmir, Turkey
(BNH-210708-1).
tions showed that 3 had the same relative configurations at C-3 (b-
OH), C-6 (a-OH) and C-16 (b-OH) positions as in 1 and 2. It has to
be mentioned that the 13C NMR data can be regarded as
characteristic parameters in the determination of absolute
3.3. Extraction and isolation
The air-dried and powdered plant material of Astragalus
schottianus Boiss (whole plant; 400 g) was extracted with MeOH
(2ꢁ 5 L) at 70 8C for 12 h, under reflux. After filtration, the solvent
was removed by rotary evaporation to yield 28.37 g of crude
extract. The MeOH extract was subjected to vacuum liquid
chromatography (VLC) on reversed-phase material (Lichroprep
RP-18, 25–40
mm, 200 g) employing H2O (350 mL), H2O–MeOH
(85:25, 1000 mL; 25:75, 600 mL), and MeOH (1300 mL) to give ten
main fractions (A1–A10). After TLC analysis, fractions A3–A10 were
found to be rich in saponins. Frs. A3–A10 (17.09 g) was combined
and applied to open column chromatography using silica gel
(500 g) as stationary phase. Elution was carried out with CHCl3–
MeOH mixtures (95:5, 3000 mL; 90:10, 400 mL; 85:15, 10,000 mL;
Fig. 2. Spin systems (bold lines) and key long-range correlations (arrows from C to
H) of 3 deduced from DQF-COSY/HSQC and HMBC spectra, respectively.

F. Karabey et al. / Phytochemistry Letters 5 (2012) 320–324
323
80:20, 3000 mL; 70:30, 2000 mL) to give fifty-nine fractions (B).
Fractions B28–B32 were pooled together and chromatographed
over a Biotage Flash Chromatography using reversed-phase 25 M
column. Elution was performed by using H2O–MeOH gradient [70%
MeOH (350 mL), 70 ! 80% (100 mL) and 80 ! 85% (100 mL)] to
give seventy-six fractions (C). Compound 1 was obtained from
fraction C60–C61 (54.2 mg). The fraction B51–B54 (716.3 mg) was
applied to Biotage Flash Chromatography using reversed-phase
25 M column. Elution was carried out with a gradient system: 100%
H2O ! 20% MeOH (80 mL), 20% ! 60% MeOH (90 mL), 60%–80%
MeOH (50 mL), and 80% MeOH (50 mL) to give ninety-three
fractions (D). Fractions D67–D70 (23.9 mg) yielded compound 2.
The fraction B18–B22 (379.7 mg) was subjected to vacuum liquid
chromatography (VLC) on reversed-phase material (Lichroprep RP-
2000), 4.05 (1H, m, H-200), 3.98 (1H, m, H-5000), 3.98 (1H, m, H-500), 3.84
(1H, brs, H-24), 3.74 (1H, m, H-6), 3.66 (1H, dd, J = 10.8, 10.0 Hz, H-
50a), 3.55 (1H, dd, J = 11.6, 4.4 Hz, H-3), 2.09 (1H, m, H-17), 1.53
(1H, s, H-21), 1.92 (1H, m, H-8), 1.69 (1H, d, J = 9.6 Hz, H-5), 0.51
and 0.12 (each 1H, d, JAB = 4.4 Hz, H-19a and H-19b, respectively);
HRESIMS [ESI(+)MicroTOF]: (m/z 981.48275 [M-Cl]ꢀ (calcd for
C47H78O20Cl).
3.3.3. 3-O-
hexahydroxycycloartane (3)
Amorphous powder; 13C-NMR (100 MHz, Pyridin-d6):
b-D-xylopyranosyl-3b,6a,16b,20(S),24(R),25-
d
107.6
(d, C-10), 88.4 (d, C-3), 79.6 (d, C-24), 78.4 (d, C-30), 76.7 (s, C-20),
75.5 (d, C-16), 73.8 (d, C-20), 72.6 (s, C-25), 71.0 (d, C-40), 67.7 (d, C-
6), 66.8 (d, C-50), 56.4 (d, C-17), 54.0 (d, C-5), 48.6 (t, C-15), 46.9 (s,
C-14), 46.5 (s, C-13), 46.4 (d, C-8), 42.5 (s, C-4), 39.8 (t, C-22), 38.4 (t,
C-7), 33.5 (t, C-12), 32.2 (t, C-1), 30.1 (t, C-2), 29.8 (s, C-10), 29.1 (q,
C-28), 28.7 (q, C-21), 28.4 (t, C-19), 26.3 (t, C-11, C-23), 26.1 (q, C-
27), 25.7 (q, C-26), 20.9 (q, C-18), 20.3 (s, C-9), 20.1 (q, C-30), 16.2 (q,
18, 25–40
mm, 25 g) employing, H2O–MeOH (75:25, 500 mL;
60:40, 600 mL), and MeOH (1300 mL) to give fifty main fractions
(E). Fraction E14–E28 (36.4 mg) was subjected to open column
chromatography using silica gel (15 g) as the stationary phase.
Elution was carried out with CHCl3–MeOH–H2O mixtures
(85:15:0.5, 500 mL; 80:20:1 400 mL) to give fifty-eight fractions
(F). Fraction F33–F58 yielded compound 3 (7.2 mg).
C-29); 1H NMR (400 MHz, Pyridin-d6):
d 4.95 (1H, m, H-16), d 4.61
(1H, d, J = 7.2 Hz, H-10), 4.38 (1H, dd, J = 5.6, 11.6 Hz, H-50a), 4.26
(1H, m, H-40), 4.18 (1H, dd, J = 8.8, 8.0 Hz, H-30), 4.09 (1H, dd, J = 8.0,
7.2 Hz, H-20), 3.93 (1H, brd, J = 10.0 Hz, H-24), 3.76 (1H, m, H-6),
3.74 (1H, m, H-50b), 3.58 (1H, dd, J = 10.4, 5.2 Hz, H-3), 2.38, (1H, m,
H-22a), 2.34 (1H, m, H-2a), 2.26 (1H, m, H-22b), 2.11 (1H, m, H-
15a), 2.11 (1H, d, J = 6.8 Hz, H-17), 2.02 (1H, m, H-12a), 2.02 (3H, s,
H-29), 1.94 (2H, m, H-2b and H-8), 1.91 (1H, m, H-23a), 1.86 (1H,
m, H-23b), 1.79 (1H, m, H-15b), 1.79 (3H, s, H-18), 1.75 (1H, m, H-
7a), 1.68 (1H, m, H-12b), 1.68 (1H, s, H-21), 1.67 (1H, m, H-5), 1.57
(3H, s, H-27), 1.55 (1H, m, H-7b), 1.54 (3H, s, H-26), 1.35 (3H, s, H-
28), 1.03 (3H, s, H-30), 0.58 and 0.28 (each 1H, d, JAB = 4.0 Hz, H-19a
and H-19b, respectively); HRESIMS [ESI(+)MicroTOF]: (m/z
675.38677) [M-Cl]ꢀ (calcd for C35H60O10Cl, 675.38750).
3.3.1. 20(R),25-epoxy-3-O-
pyranosyl-3 ,6 ,16 ,24 -tetrahydroxycycloartane (1)
Amorphous powder; 13C NMR (100 MHz, Pyridin-d6):
b-D-xylopyranosyl-24-O-b-D-gluco-
b
a
b
a
d
C 108.1
(d, C-10), 101.3 (d, C-100), 89.2 (d, C-3), 79.9 (s, C-20), 79.1 (d, C-30),
79.0 (d, C-500), 78.8 (d, C-300), 76.2 (d, C-20), 75.8 (d, C-200), 75.5 (s, C-
25), 74.8 (d, C-16), 74.7 (d, C-24), 74.1 (d, C-400), 71.8 (d, C-40), 68.7
(d, C-6), 67.6 (d, C-50), 63.5 (t, C-600), 61.3 (d, C-17), 54.7 (d, C-5), 47.8
(d, C-8), 47.7 (s, C-13), 47.2 (s, C-4), 46.5 (t, C-15), 43.3 (s, C-10),
39.3 (t, C-7), 34.4 (s, C-14), 33.0 (t, C-2), 30.9 (t, C-12), 30.0 (t, C-1),
29.5 (t, C-19), 29.3 (q, C-26), 29.0 (q, C-28), 28.9 (q, C-27), 28.6 (q, C-
21), 27.6 (t, C-22), 26.9 (t, C-11), 21.8 (t, C-23), 21.5 (q, C-18), 20.8
(q, C-30), 19.5 (s, C-9), 17.2 (q, C-29). 1H NMR (400 MHz, Pyridin-
3.3.4. Acid hydrolysis of compounds 1–3
d6):
d
4.90 (1H, d, J = 7.2 Hz, H-10), 4.88 (1H, d, J = 7.6 Hz, H-100), 4.82
A solution of each compound (1.0 mg) in 2 N HCl (1 mL) was
(1H, dd, H-16), 4.56 (1H, dd, J = 12.2, 2.0 Hz, H-600a), 4.36 (1H, dd,
J = 11.2, 5.2 Hz, H-50a), 4.35 (1H, dd, H-500b), 4.18 (1H, m, H-40), 4.15
(1H, t, J = 8.4 Hz, H-30), 4.21 (1H, m, H-300), 4.16 (1H, m, H-400), 3.96
(1H, m, H-500), 4.04 (1H, m, H-200), 4.06 (1H, m, H-20), 3.84 (1H, brs,
H-24), 3.74 (1H, m, H-6), 3.71 (1H, y, H-50b), 3.61 (1H, m, H-3), 1.96
(1H, m, H-17), 1.80 (1H, dd, J = 12.0, 4.0 Hz, H-8), 1.53 (1H, s, H-21),
1.72 (1H, d, J = 9.2 Hz, H-5), 0.12 and 0.49 (each 1H, d, JAB = 4.0 Hz,
H-19a and H-19b, respectively); HRESIMS [ESI(+)MicroTOF]: (m/z
807.46042) [M+Na]+ (calcd for C41H68O14Na, 807.45068).
refluxed for 3 h. Under a stream of N2, the solution was
concentrated. The residue was dissolved in 1-(trimethylsilyl)-
imidazole and pyridine (0.1 ml), and the solution was stirred at
60 8C for 5 min. After drying under a stream of N2, the residue was
partitioned between H2O and CHCl3 (1 ml, 1:1 v/v). The CHCl3 layer
was analyzed by gas chromatography with an L-Chirasil-Val
column (0.32 mm ꢁ 25 m). Temperatures of the injector and
detector were 200 8C. A temperature program was used for the
oven, starting at 100 8C for 1 min and increasing up to 180 8C at a
rate of 5 8C/min. The chromatographic peaks of the hydrolysate of 1
3.3.2. 20(R),25-epoxy-3-O-[
xylopyranosyl-24-O- -glucopyranosyl-3
tetrahydroxycycloartane (2)
Amorphous powder; 13C NMR: 981.48258. 13C NMR (100 MHz,
Pyridin-d6)
106.0 (d, C-100), 105.6 (d, C-10), 100.7 (d, C-1000), 88.6 (d,
b
-
D
-glucopyranosyl(1!2)]-
b
-
D
-
and 2 were detected at 14.64 and 14.72 min, respectively, for
glucose, and 10.96/12.02 and 10.99/12.04 min, respectively for
xylose. The peaks of D-xylose (10.96 and 12.01 min) were detected
in the hydrolysate of 3. Retention times for authentic samples were
D
-
-
b
-D
b
,6 ,16 , 24
a
b
a
-
D
d
found at 14.71 min (
xylose).
D-glucose), and 10.98 and 12.01 min (D-
C-3), 83.3 (d, C-20), 79.4 (s, C-20), 78.6 (d, C-3000), 78.5 (d, C-300), 77.9
(d, C-500), 77.8 (d, C-5000, C-30), 76.9 (d, C-2000), 75.2 (d, C-200), 74.9 (s, C-
25), 74.6 (d, C-24), 74.1 (d, C-16), 71.8 (d, C-4000), 71.7 (d, C-400), 70.9
(d, C-40), 67.8 (d, C-6), 66.6 (d, C-50), 63.0 (t, C-600), 62.7 (t, C-6000),
60.8 (d, C-17), 54.0 (d, C-5), 47.2 (t, C-15), 46.7 (d, C-8), 46.6 (s, C-
14), 45.9 (s, C-13), 42.8 (s, C-4), 38.6 (t, C-7), 33.9 (t, C-12), 32.3 (t, C-
1), 30.5 (t, C-19), 29.8 (s, C-10), 29.3 (t, C-2), 29.3 (q, C-28), 28.6 (q,
C-26), 28.0 (q, C-21), 26.8 (q, C-27), 26.3 (t, C-22), 22.9 (t, C-11),
21.2 (t, C-23), 20.9 (s, C-9), 20.2 (q, C-18), 18.9 (q, C-30), 16.5 (q, C-
Acknowledgments
We wish to thank Dr. Markus Ganzera for his assistance with
the HR-MS experiments and Dr. H. As¸ kın Akpulat for authentica-
tion of the plant species. We also acknowledge partial financial
support from the Global Research Network for Medicinal Plants
(GRNMP) and King Saud University.
29); 1H NMR (400 MHz, Pyridin-d6)
d
5.40 (1H, d, J = 7.8 Hz, H-100),
4.92 (1H, d, J = 7.2 Hz, H-10), 4.90 (1H, d, J = 8.0 Hz, H-1000), 4.81 (1H,
m, H-16), 4.60 (1H, dd, J = 12.4, 2.0 Hz, H-6000a), 4.52 (1H, dd,
J = 11.6, 3.2 Hz, H-600a), 4.47 (1H, dd, J = 12.0, 3.6 Hz, H-600b), 4.38
(1H, dd, J = 12.4, 5.6 Hz, H-6000b), 4.32 (1H, m, H-400), 4.26 (1H, m, H-
30), 4.26 (1H, m, H-50a), 4.25 (1H, m, H-300), 4.23 (1H, m, H-20), 4.21
(1H, m, H-3000), 4.20 (1H, m, H-40), 4.18 (1H, m, H-4000), 4.14 (1H, t, H-
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytol.2012.02.011.

324
F. Karabey et al. / Phytochemistry Letters 5 (2012) 320–324
¨
¨
Gu¨lcemal, D., Alankus¸-C¸alıs¸kan, O., Perrone, A., Ozgo¨kc¸e, F., Piacente, S., Bedir, E., 2011.
Cycloartane glycosides from Astragalus aureus. Phytochemistry 72, 761–768.
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