Triterpene glycosides from astragalus angustifolius

Article abstract of DOI:10.1055/s-0031-1298337

Six new cycloartane-type (1-6) and four new oleanane-type (7-10) triterpene glycosides were isolated from Astragalus angustifolius Lam., together with five known triterpene glycosides. Their structures were established by the extensive use of 1D and 2D-NMR experiments along with ESIMS and HRMS analysis. Compounds 1-3 are glycosides of cycloastragenol, while compounds 4-6 show the C-24 epimer of cycloastragenol as aglycone, encountered for the first time in nature. All compounds were evaluated for their antiproliferative activity in Hela, H-446, HT-29, and U937 cell lines. Only compound 8 displayed a weak activity with IC₅₀ values of 36 and 50 μM against Hela and HT-29 cell lines, respectively. Georg Thieme Verlag KG Stuttgart · New York.

Full text of DOI:10.1055/s-0031-1298337

720 Original Papers
Triterpene Glycosides from Astragalus angustifolius
Authors
Derya Gülcemal1, Milena Masullo2, Erdal Bedir3, Michela Festa2, Tamer Karayıldırım1, Ozgen Alankus-Caliskan1,
Sonia Piacente2
1
Affiliations
Department of Chemistry, Faculty of Science, Ege University, Bornova, Izmir, Turkey
Dipartimento di Scienze Farmaceutiche e Biomediche, Università degli Studi di Salerno, Fisciano, Salerno, Italy
Department of Bioengineering, Faculty of Engineering, Ege University, Bornova, İzmir, Turkey
2
3
Key words
Abstract
genol as aglycone, encountered for the first time
in nature. All compounds were evaluated for their
antiproliferative activity in Hela, H-446, HT-29,
and U937 cell lines. Only compound 8 displayed
a weak activity with IC50 values of 36 and 50 µM
against Hela and HT-29 cell lines, respectively.
"
l Astragalus angustifolius
!
"
l Leguminosae
Six new cycloartane-type (1–6) and four new ole-
anane-type (7–10) triterpene glycosides were
isolated from Astragalus angustifolius Lam., to-
gether with five known triterpene glycosides.
Their structures were established by the exten-
sive use of 1D and 2D‑NMR experiments along
with ESIMS and HRMS analysis. Compounds 1–3
are glycosides of cycloastragenol, while com-
pounds 4–6 show the C-24 epimer of cycloastra-
"
l cycloartane saponins
"
l oleanane saponins
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
Introduction
!
and four new oleanane-type triterpene glycosides
(7–10; l Fig. 2) from the methanol extract of
"
Astragalus L. (Leguminosae) is the genus compris-
ing the highest number of species among the
spermatophytes. The exact number of species in
this genus has not been established yet, but it is
estimated to be around 3000 [1]. In Turkey it is
represented by 445 species, of which 224 are en-
demic [2]. Earlier investigations on Turkish Astra-
galus species resulted in the isolation of a series of
oleanane- and cycloartane-type saponins [3–11].
Previous studies have shown interesting biologi-
cal activities, including immunostimulating [7,
12], anti-protozoal [13], antiviral [14], cytotoxic
[15], cardiotonic [16], wound healing [17], and
adjuvant properties [18] for cycloartane- and ole-
anane-type glycosides isolated from Astragalus
species. In particular, cycloartane glycosides
showed antiproliferative activity against several
cancer lines including solid tumor (HepG2), blood
tumor (HL-60), and drug resistant tumor
(R‑HepG2) [19].
A. angustifolius, along with three known cycloar-
tane glycosides (11–13) and two known oleanane
glycosides (14, 15).
The antiproliferative activity of compounds 1–15
was tested in different cancer cell lines including
Hela, H-446, HT-29, and U937. Only compound 8
displayed a weak activity with an IC50 of 36 and
50 µM against Hela and HT-29 cell lines, respec-
tively.
received
revised
accepted
Dec. 14, 2011
February 7, 2012
February 13, 2012
Bibliography
Materials and Methods
!
DOI http://dx.doi.org/
10.1055/s-0031-1298337
Published online March 21,
2012
Planta Med 2012; 78: 720–729
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 0032‑0943
General
Optical rotations were measured on a JASCO DIP
1000 polarimeter. IR measurements were ob-
tained on a Bruker IFS-48 spectrometer. NMR ex-
periments were performed on a Bruker DRX-600
spectrometer (Bruker BioSpinGmBH) equipped
with a Bruker 5-mm TCI CryoProbeat 300 K. All
2D‑NMR spectra were acquired in CD3OD
(99.95%, SigmaAldrich) and standard pulse se-
quences and phase cycling were used for DQF-
COSY, HSQC, and HMBC spectra. The NMR data
were processed using UXNMR software. Exact
masses were measured by a Voyager DE mass
spectrometer. Samples were analyzed by matrix-
Correspondence
Prof. Dr. Sonia Piacente
Dipartimento di Scienze Farma-
ceutiche e Biomediche
Università degli Studi di Salerno
Via Ponte Don Melillo
84084 Fisciano, Salerno
Italy
Phone: + 3989969763
Fax: + 3989969602
piacente@unisa.it
Continuing our studies on the constituents of
Astragalus species, the investigation of the whole
parts of Astragalus angustifolius Lam., used as an
anti-inflammatory remedy in Turkish folk medi-
cine [2], was carried out. Here we report the iso-
lation and structure elucidation of six new cyclo-
"
artane-type triterpene glycosides (1–6; l Fig. 1)
Gülcemal D et al. Triterpene Glycosides from… Planta Med 2012; 78: 720–729

Original Papers 721
Fig. 1 Chemical structures of compounds 1–6.
assisted laser desorption ionization time-of-flight (MALDITOF)
mass spectrometry. A mixture of analyte solution and α-cya-
no-4-hydroxycinnamic acid (Sigma) was applied to the metal-
lic sample plate and dried. Mass calibration was performed
with the ions from ACTH (fragment 18–39) at 2465.1989 Da and
α-cyano-4-hydroxycinnamic acid at 190.0504 Da as the internal
standard. ESIMS analyses were performed using a ThermoFin-
nigan LCQ Deca XP Max iontrap mass spectrometer equipped
with Xcalibur software. GC analysis was performed on a Termo
Gülcemal D et al. Triterpene Glycosides from… Planta Med 2012; 78: 720–729

722 Original Papers
Fig. 2 Chemical structures of compounds 7–10.
Finnigan Trace GC apparatus using a l-Chirasil-Val column
(0.32 mm × 25 m).
sity, Izmir, Turkey). A voucher specimen has been deposited in
the Herbarium of Ege University, Izmir, Turkey (EGE 40790).
Plant material
Extraction and isolation
Astragalus angustifolius Lam. (whole plant) was collected at Çaltili
Village, Çingi hill, from an altitude of 1200 m, Hafik-Sivas, Turkey
in July 2009. Samples of plant material were identified by Serdar
G. Senol (Deparment of Biology, Faculty of Sciences, Ege Univer-
Air-dried and powdered plant material of Astragalus angustifolius
(whole plant, 367 g) was extracted with MeOH (3.5 L × 2) at 60°C.
The methanolic solution was then evaporated to dryness under
reduced pressure and gave 28 g of a dark residue. This residue
Gülcemal D et al. Triterpene Glycosides from… Planta Med 2012; 78: 720–729

Original Papers 723
Table 1 13C and 1H NMR data (J in Hz) of the sugar portions of compounds 1, 5, and 6 (600 Mz, δ ppm, in CD3OD).
1a
5
6
δC
δ
H (J in Hz)
δC
δ
H (J in Hz)
δC
δH (J in Hz)
β-D‑Glc (at C-3)
105.7
β-D‑Xyl (at C-3)
107.8
α-L‑Rha (at C-6)
1
2
3
4
5
4.44, d (7.5)
4.28, d (7.5)
104.2
72.6
70.1
73.8
72.3
4.80, d (1.2)
79.7
3.45, dd (7.5, 9.0)
3.48, t (9.0)
75.5
3.21, dd (7.5, 9.2)
3.31, t (9.2)
3.86, dd (1.2, 3.2)
3.71, dd (3.2, 9.7)
3.42, t (9.7)
79.7
77.7
72.2
3.31, t (9.0)
71.4
3.49, m
77.7
3.26, ddd (3.5, 4.5, 9.0)
66.7
3.84, dd (5.2, 11.7)
3.19, t (11.7)
3.61, m
6
62.6
3.88, dd (3.5, 12)
3.69, dd (4.5, 12)
17.5
1.26, d (6.5)
α-L‑Rha (at C-2glc
)
α-L‑Rha (at C-6)
104.2
72.5
1
2
3
4
5
6
102.0
72.0
72.0
74.2
70.0
17.6
5.42, d (1.2)
3.99, dd (1.2, 3.2)
3.79, dd (3.2, 9.7)
3.42, t (9.7)
4.78, d (1.2)
3.86, dd (1.2, 3.2)
3.71, dd (3.2, 9.7)
3.41, t (9.7)
70.0
73.7
4.01, m
72.3
3.61, m
1.23, d (6.5)
17.6
1.27, d (6.5)
a The chemical shift values of the sugar portion of 2–4 were superimposable with those reported for 1
was suspended in H2O (350 mL), and successively partitioned
with n-hexane (200 mL × 2), EtOAc (200 mL × 2), and n-BuOH sat-
urated with H2O (200 mL × 3). The n-BuOH extract (7.7 g) was
subjected to vacuum liquid chromatography (VLC) using re-
versed-phase material (Lichroprep RP-18, 25–40 µm, 150 g,
50 cm × 5 cm) employing H2O (1000 mL), H2O–MeOH (8:2,
1200 mL; 6:4, 2400 mL; 4:6, 3000 mL; 2:8, 1600 mL) and MeOH
(600 mL) to give ten main fractions (A–J). Fraction A (780 mg) was
submitted to silica gel (100 g) column chromatography with the
solvent system CHCl3-MeOH‑H2O (80:20:2, 1500 mL; 70:30:3,
2000 mL, 150 cm × 1.5 cm) to give 7 subfractions. Subfraction 1
(120 mg) was chromatographed on a reversed-phase material
(Lichroprep RP-18, 25–40 µm, 25 g, 50 cm × 3 cm), employing
MeOH‑H2O (8:2, 500 mL) yielding 8 (15 mg) and 13 (9 mg). Sub-
fraction 2 (58 mg) was purified on a reversed-phase column (Li-
chroprep RP-18, 25–40 µm, 25 g, 50 cm × 3 cm) and eluted with
MeOH‑H2O (6:4, 500 mL) to give 7 (1.1 mg). Fraction B (300 mg)
was subjected to silica gel (42 g) column chromatography with
the solvent system EtOAc-MeOH‑H2O (100:17.5:13.5, 800 mL,
60 cm × 1.5 cm) to yield 14 (10 mg) and 9 (10 mg). Fraction D
(240 mg) was submitted to silica gel (42 g, 60 cm × 1.5 cm) col-
umn chromatography, eluted with CHCl3-MeOH‑H2O (80:20:2,
1500 mL; 70:30:3, 2000 mL) to give 6 (2 mg), 11 (1 mg), 10
(1.2 mg), 5 (23 mg), and 12 (9 mg). Fraction E (60 mg) was sub-
jected to silica gel (38 g) column chromatography with the sol-
vent system CHCl3-MeOH‑H2O (90:10:1, 600 mL; 80:20:2,
1500 mL, 100 cm × 1.5 cm) to yield 3 (9 mg) and 1 (7 mg). Fraction
D (160 mg) was submitted to silica gel (38 g) column chromatog-
raphy with the solvent system CHCl3-MeOH‑H2O (90:10:1,
100 mL; 80:20:2, 1500 mL; 70:30:3, 2000 mL, 60 cm × 1.5 cm)
to give 4 (4 mg), 2 (12 mg), and 15 (10 mg).
Compound 2: Amorphous white solid; [α]2D5 + 41.4 (c 0.1 MeOH);
IR (KBr) νmax 3480, 3035, 2945, 1260, and 1055 cm−1 1H NMR
;
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data of the
1
sugar portion are superimposable with those reported for 1; H
NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data of
"
the aglycone moiety, see l Table 2; ESI MS (pos. ione mode) m/z
895 [M + Na]+, MS/MS m/z 819 [M + Na-76]+, m/z 673 [M + Na-
76-146]+, m/z 493 [M+Na-76-146-180]+; HRMALDITOFMS m/z
895.4669 [M + Na]+ (calcd. for C44H72O17Na, 895.4667).
Compound 3: Amorphous white solid; C42H68O14; [α]2D5 − 23.1 (c
0.1 MeOH); IR (KBr) νmax 3475, 3030, 2948, 1740, 1258, and
1050 cm−1 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD,
;
150 MHz) data of the sugar portion are superimposable with
those reported for 1; 1H NMR (CD3OD, 600 MHz) and 13C NMR
"
(CD3OD, 150 MHz) data of the aglycone moiety, see l Table 2;
ESI‑MS (pos. ione mode) m/z 819 [M+Na]+, MS/MS m/z 673 [M +
Na-146]+, m/z 493 [M + Na-146-180]; HRMALDITOFMS m/z
819.4512 [M + Na]+ (calcd. for C42H68O14Na, 819.4507).
Compound 4: Amorphous white solid; [α]2D5 − 5.21 (c 0.1 MeOH);
IR (KBr) νmax 3460, 3038, 2935, 1250, and 1050 cm−1 1H NMR
;
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data of the
1
sugar portion are superimposable with those reported for 1; H
NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data of
"
the aglycone moiety, see l Table 2; ESI‑MS (pos. ione mode) m/z
821 [M + Na]+, MS/MS m/z 675 [M + Na-146]+, m/z 495 [M + Na-
146-180]+; HRMALDITOFMS m/z 821.4666 [M + Na]+ (calcd. for
C
42H70O14Na, 821.4663).
Compound 5: Amorphous white solid; [α]2D5 + 15.1 (c 0.1 MeOH);
IR (KBr) νmax 3460, 3035, 2940, 1240, and 1050 cm−1 1H NMR
;
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data of the
sugar portion, see l Table 1; H NMR (CD3OD, 600 MHz) and 13C
1
"
Compound 1: Amorphous white solid; [α]2D5 + 19.01 (c 0.1 MeOH);
NMR (CD3OD, 150 MHz) data of the aglycone moiety, see l Table
"
IR (KBr) νmax 3470, 3040, 2950, 1260, and 1058 cm−1
;
1H NMR
2; ESI‑MS (pos. ione mode) m/z 791 [M + Na]+, MS/MS m/z 645
[M + Na-146]+, m/z 495 [M + Na-146-150]+; HRMALDITOFMS
m/z 791.4561 [M + Na]+ (calcd. for C41H68O13Na, 791.4558).
Compound 6: Amorphous white solid; [α]2D5 − 8.09 (c 0.1 MeOH);
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data of the
sugar portion, see l Table 1; 1H NMR (CD3OD, 600 MHz) and
"
13C NMR (CD3OD, 150 MHz) data of the aglycone moiety, see
+
l Table 2; ESI‑MS (pos. ione mode) m/z 879 [M+Na] ; MS/MS
m/z 803 [M + Na-76]+, m/z 657 [M + Na-76-146]+, 477 [M + Na-
76-146-180]+; HRMALDITOFMS m/z 879.4722 [M+Na]+ (calcd.
for C44H72O16Na, 879.4718).
IR (KBr) νmax 3450, 3042, 2938, 1250, and 1052 cm−1 1H NMR
;
"
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data of the
1
sugar portion, see l Table 1; H NMR (CD3OD, 600 MHz) and 13C
"
"
NMR (CD3OD, 150 MHz) data of the aglycone moiety, see l Table
Gülcemal D et al. Triterpene Glycosides from… Planta Med 2012; 78: 720–729

724 Original Papers
Gülcemal D et al. Triterpene Glycosides from… Planta Med 2012; 78: 720–729

Original Papers 725
Table 3 13C and 1H NMR data (J in Hz) of the sugar portions of compounds 7, 9, and 10 (600 Mz, δ ppm, in CD3OD).
ꢀ7a
ꢀδC
ꢀ9
ꢀ10
ꢀδC
δ
H (J in Hz)
ꢀδC
δ
H (J in Hz)
δH (J in Hz)
β-D-GlcA (at C-3)
β-D-GlcA (at C-3)
β-D‑Glc (at C-3)
1
2
3
4
5
6
105.6
78.8
78.8
74.1
77.0
177.1
4.44, d (7.5)
105.6
78.6
78.6
74.2
76.9
177.2
4.43, d (7.5)
105.2
75.3
77.9
71.6
78.3
62.8
4.26, d (7.5)
3.22, dd (7.5, 9.0)
3.58, dd (7.5, 9.0)
3.65, dd (9.0, 9.0)
3.43, dd (9.0, 9.0)
3.57, d (9.0)
–
3.58, dd (7.5, 9.0)
3.64, dd (9.0, 9.0)
3.43,dd (9.0, 9.0)
3.57, d (9.0)
–
3.28, dd (9.0, 9.0)
3.29, dd (9.0, 9.0)
3.38, ddd (3.5, 4.5, 9.0)
3.90, dd (3.5, 12)
3.69, dd (4.5, 12)
β-D‑Xyl (at C-2glcA
)
β-D‑Xyl (at C-2glcA)
1
2
3
4
5
103.3
79.3
77.4
71.0
66.6
4.82, d (7.5)
103,2
79.4
76.5
71.0
66.6
4.82, d (7.5)
3.41, dd (7.5, 9.2)
3.52, dd (9.2, 9.2)
3.53, m
3.40, dd (7.5, 9.2)
3.57, dd (9.2, 9.2)
3.54, m
3.83, dd (5.2, 11.7) 3.11, t (11.7)
3.84, dd (5.2, 11.7)
3.11, t (11.7)
6
α-L‑Rha (at C-2xyl
)
α-L‑Rha (at C-2xyl)
1
2
3
4
5
6
102.3
72.3
72.2
74.1
69.7
17.6
5.23, d (1.2)
3.96, dd (1.2, 3.2)
3.76, dd (3.2, 9.7)
3.42, t (9.7)
102.3
72.0
72.1
74.2
69.7
17.5
5.23, d (1.2)
3.97, dd (1.2, 3.2)
3.78, dd (3.2, 9.7)
3.43, t (9.7)
4.15, m
4.16, m
1.26, d (6.5)
1.25, d (6.5)
α-L‑Ara (at C-22)
1
2
3
4
5
102.0
74.2
72.2
69.0
65.7
4.27, d (3.7)
3.60, dd (8.5, 3.7)
3.58, dd (8.5, 3.0)
3.86, m
3.89 dd (11.9, 2.0)
3.52, dd (11.9, 3.0)
a The chemical shift values of the sugar portion of 8 were superimposable with those reported for 7
2; ESI‑MS (pos. ione mode) m/z 659 [M + Na]+, MS/MS m/z 513
[M + Na-146]+; HRMALDITOFMS m/z 659.4139 [M + Na]+ (calcd.
for C36H60O9Na, 659.4135).
[M + Na-146-132-176]+; HRMALDITOFMS m/z 1067.5406 [M +
Na]+ (calcd. for C52H84O21Na, 1067.5403).
Compound 10: Amorphous white solid; [α]2D5 + 27.7 (c 0.1 MeOH);
Compound 7: Amorphous white solid; [α]2D5 − 10.9 (c 0.1 MeOH);
IR (KBr) νmax 3440, 2933, 1645, 1240, and 1044 cm−1 1H NMR
;
IR (KBr) νmax 3455, 2945, 1666, 1252, and 1052 cm−1
;
1H NMR
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data of the
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data of the
sugar portion, see l Table 3; H NMR (CD3OD, 600 MHz) and 13C
1
"
1
sugar portion, see l Table 3; H NMR (CD3OD, 600 MHz) and 13C
NMR (CD3OD, 150 MHz) data of the aglycone moiety, see l Table
"
"
NMR (CD3OD, 150 MHz) data of the aglycone moiety, see l Table
4; ESI‑MS (pos. ione mode) m/z 659 [M + Na]+, MS/MS m/z 439
[M + Na-180]+; HRMALDITOFMS m/z 659.4139 [M + Na]+ (calcd.
for C36H60O9Na, 659.4135).
"
4; ESI‑MS (pos. ione mode) m/z 967 [M + Na]+, MS/MS m/z 821
[M + Na-146]+, m/z 689 [M + Na-146-132]; HRMALDITOFMS m/
z 967.4881 [M + Na]+ (calcd. for C47H76O19Na, 967.4879).
Compound 8: Amorphous white solid; [α]2D5 + 7.12 (c 0.1 MeOH);
Acid hydrolysis
IR (KBr) νmax 3451, 2937, 1735, 1658, 1248, and 1048 cm−1
;
1H
The configurations of the sugar units were established after hy-
drolysis of 1–10 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. [20].
The peaks of the hydrolysate of 1 were detected at 10.72 (L-rham-
nose) and at 14.73 min (D-glucose). The peaks of the hydrolysate
of 5 were detected at 10.73 (L-rhamnose) and 10.98 and 12.02 (D-
xylose). The peaks of L-arabinose (8.94 and 9.81 min), L-rham-
nose (10.72), D-xylose (10.98 and 12.01 min), and D-glucuronic
acid (15.83 min) were detected in the hydrolysate of 9. For the
hydrolysate of 10 a peak at 14.73 min (D-glucose) was detected.
Retention times for authentic samples after being treated in the
same manner with 1-(trimethylsilyl)-imidazole in pyridine were
detected at 8.92 and 9.80 (L-arabinose), 10.70 (L-rhamnose),
NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data of
the sugar portion are superimposable with those reported for 7;
1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data
"
of the aglycone moiety, see l Table 4; ESI‑MS (pos. ione mode) m/
z 965 [M + Na]+, MS/MS m/z 819 [M + Na-146]+, m/z 687 [M + Na-
146-132]; HRMALDITOFMS m/z 965.4726 [M + Na]+ (calcd. for
C
47H74O19Na, 965.4722).
Compound 9: Amorphous white solid; [α]2D5 − 8.54 (c 0.1 MeOH);
IR (KBr) νmax 3446, 2930, 1730, 1648, 1245, and 1052 cm−1 1H
;
NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data of
1
"
the sugar portion, see l Table 3; H NMR (CD3OD, 600 MHz) and
13C NMR (CD3OD, 150 MHz) data of the aglycone moiety, see
+
"
l Table 3; ESI‑MS (pos. ione mode) m/z 1067 [M + Na] , MS/MS
m/z 921 [M + Na-146]+, m/z 789 [M + Na-146-132], m/z 613
Gülcemal D et al. Triterpene Glycosides from… Planta Med 2012; 78: 720–729

726 Original Papers
Table 4 13C and 1H NMR data (J in Hz) of the aglycone moieties of compounds 7–10 (600 Mz, δ ppm, in CD3OD).
ꢀ7
ꢀ8
ꢀ9
ꢀ10
ꢀδC
ꢀδC
δ
H (J in Hz)
ꢀδC
δ
H (J in Hz)
ꢀδC
δ
H (J in Hz)
δH (J in Hz)
1
2
39.7
1.64, 1.04, m
2.18, 1.80, m
3.35, dd (11.5, 4.5)
–
39.7
1.66, 1.04, m
2.18, 1.80, m
3.36, dd (11.5, 4.2)
–
39.5
1.67, 1.04, m
2.18, 1.82, m
3.35, dd (11.5, 4.2)
–
39.8
1.71, 1.01, m
2.18, 1.82, m
3.36, dd (11.5, 4.2)
–
26.7
92.4
44.0
57.5
19.0
33.8
41.0
48.7
37.5
24.6
123.7
146.0
43.1
27.3
30.4
39.7
43.9
41.3
41.5
70.7
80.8
22.8
64.2
26.8
92.6
44.4
57.5
19.1
34.7
40.4
48.7
37.5
24.5
124.1
145.5
43.5
26.3
29.9
38.2
46.4
42.8
44.4
38.8
77.3
22.6
63.6
26.8
91.9
44.4
57.0
19.0
33.7
40.0
48.3
36.7
24.5
123.8
145.1
42.5
26.1
29.2
36.8
46.9
47.0
31.1
36.8
83.3
22.8
63.4
28.4
81.6
43.8
57.3
19.4
34.8
40.5
49.0
37.6
24.5
124.1
145.4
43.0
26.5
29.9
38.4
45.9
42.0
36.2
36.8
77.1
23.0
65.3
3
4
5
0.97, m
0.98, m
0.96, m
0.89, m
6
1.67, 1.41
1.61,1.38, m
–
1.66, 1.39, m
1.60,1.43, m
–
1.67, 1.41, m
1.60,1.43, m
–
1.68, 1.42, m
1.56, 1.43, m
–
7
8
9
1.62, m
1.60, m
1.60, m
1.59, m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
–
–
–
–
1.92, (2H) m
5.27, t (3.5)
–
1.90, (2H) m
5.31, t (3.5)
–
1.90, (2H) m
5.31, t (3.5)
–
1.91, (2H) m
5.31, t (3.5)
–
–
–
–
–
1.90, 1.02, m
1.91, 1.00, m
–
1.80, 1.06, m
1.80, 1.31, m
–
1.79, 1.05, m
1.80, 1.38, m
–
1.78, 1.06, m
1.78, 1.39, m
–
2.29, m
2.02, m
2.12, m
2.12, m
2.12, 0.99, m
–
2.38, 1.18, m
–
1.78, 1.00, m
–
1.89, 0.90, m
–
3.83, brs
3.36, d (3.5)
1.24, s
2.08, 1.45, m
3.52, brs
1.24, s
1.57, 1.37, m
3.44, brs
1.25, s
1.72, 1.34, m
3.50, brs
1.24, s
4.12, d (11.5)
3.23, d (11.5)
0.90, s
4.11, d (11.4)
3.23, d (11.4)
0.91, s
4.12, d (11.4)
3.23, d (11.4)
0.90, s
4.14, d (11.4)
3.41, d (11.4)
0.99, m
25
26
27
28
29
15.9
17.2
26.8
21.9
71.8
15.9
17.5
24.9
19.8
26.2
16.0
17.5
25.4
21.1
32.5
16.6
17.1
25.0
19.9
80.2
1.00, s
1.00, s
1.00, s
1.01, m
1.23, s
1.18, s
1.16, s
1.15, m
0.98, s
0.83, s
0.93, s
0.85, m
3.45, d (10.5)
3.12, d (10.5)
1.03
1.24, s
0.94, s
3.68, d (9.7)
3.17, d (9.7)
1.08, s
30
16.8
187.8
–
28.8
1.06, s
24.4
10.98 and 12.00 min (D-xylose), 14.71 min (D-glucose), and
15.81 min (D-glucuronic acid).
Supporting information
NMR data for the new compounds 1–10 are available as Support-
ing Information.
Cancer cell lines
Human cervical (Hela), human lung (H-446), and human colon
(HT-29) cancer cells, obtained from the European Collection of
Cell Cultures, were cultured in DMEM medium supplemented
with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum
(FBS), and 1% penicillin/streptomycin (all from Cambrex Bio-
science); human leukemic monocyte lymphoma (U937) cells
were cultured in RPMI medium (Cambrex Bioscience) supple-
mented with 2 mM L-glutamine, 10% FBS, and 1% penicillin/
streptomycin at 37°C in an atmosphere of 95% O2 and 5% CO2.
The cells were used up to a maximum of 10 passages.
Results and Discussion
!
A detailed comparison of the NMR (1H, 13C, 1D-TOCSY, HSQC,
HMBC, DQF-COSY) and ESIMS data of the sugar portion of com-
pounds 1–4 showed that the four compounds possessed the same
disaccharide chain. In particular, the H NMR spectrum of com-
pound 1 exhibited two anomeric proton doublets at δ 4.44
1
"
(J = 7.5 Hz) and 5.42 (J = 1.2 Hz) (l Table 1). In the HSQC spectrum,
these protons correlated to carbons at δ 105.7 and 102.0, respec-
tively (l Table 1). Complete assignments of the H and 13C NMR
1
"
MTT bioassay
signals allowed us the identification of an α-rhamnopyranosyl (δ
5.42) unit and a β-glucopyranosyl (δ 4.44) unit. The glycosidation
site on the aglycone of 1 as well as the position of the interglyco-
sidic linkage were determined by HMBC experiment, which
showed long-range correlations between the anomeric proton
signal at δ 4.44 (Η-1glc) and the carbon resonance at δ 89.9 (C-3),
and between the anomeric proton signal at δ 5.42 (Η-1rha) and the
carbon resonance at δ 79.7 (C-2glc). The configurations of glucose
and rhamnose units were established as D for glucose unit and L for
rhamnoseunitafterhydrolysis of 1with 1 NHCl, trimethylsilation,
and determination of retention time by GC [20]. Thus, the sugar
Human cancer cells (3 × 103) were plated in 96-well culture plates
in 90 µL of culture medium and incubated at 37°C in humidified
5% CO2. The next day, 10 µL aliquots of serial dilutions of each test
compound (purity > 97%, 1–50 µM) were added to the cells and
incubated for 48 h. Etoposide (purity = 98%) purchased from Sig-
ma was used as the positive control. Cell viability was assessed
through the MTT assay as previously reported [21].
The optical density (OD) of each well was measured with a mi-
croplate spectrophotometer (Titertek Multiskan MCC/340)
equipped with a 620-nm filter.
Gülcemal D et al. Triterpene Glycosides from… Planta Med 2012; 78: 720–729

Original Papers 727
sequence of compounds 1–4 was established as 3-O-[α-L-rham-
nopyranosyl-(1 → 2)-β-D-glucopyranosyl].
of C-20 and C-24 were derived from the ROESY spectrum, which
showed key correlation peaks between Me-18 (δ 1.44) and H-23β
(δ 1.85), which in turn correlated with H-24 (δ 3.84) suggesting a
β orientation for H-24. Further ROESY correlations between Η-17
(δ 2.27), Me-21 (δ 1.36), and Me-30 (δ 0.99) were observed. These
dataconfirmedthat theaglyconeof4 wastheC-24 epimerofcyclo-
astragenol. 20,24-Epoxycycloartanes represent the largest group
belonging to the class of cycloartane triterpenoids. Of the four pos-
sible side-chain stereoisomers [20R,24S, 20S,24R, 20R,24R and
20S,24S], to the authorsʼ knowledge, at present only the first two
have been found in Astragalus spp. [24]. Therefore, this is the first
report of a 20,24-epoxycycloartane with a 20(R),24(R) configura-
tion. On the basis of these data, the structure of the new com-
pound 4 was established as 3-O-[α-L-rhamnopyranosyl-(1 → 2)-
β-D-glucopyranosyl]-3β,6α,16β,25-tetrahydroxy-20(R),24(R)-
The ESIMS spectrum of compound 1 showed the [M + Na]+ ion at
m/z 879. The MS/MS spectrum of this ion showed peaks at m/z
803 [M + Na-76]+, due to the loss of a hydroxyacetate unit, at m/
z 657 [M + Na-76-146]+, corresponding to the subsequent loss of
a deoxyhexose unit and at m/z 477 [M + Na-76-146-180]+, as-
cribable to the loss of a hexose unit.
The 1H NMR spectrum showed for the aglycone moiety signals
due to a cyclopropane methylene at δ 0.58 and 0.39 (each 1H, d,
J = 4.8 Hz), seven tertiary methyl groups at δ 1.36, 1.33 (6H), 1.16,
1.14, 1.06, 1.05 and four methine protons at δ 5.49 (ddd, J = 8.0,
8.0, 5.6 Hz), 3.77 (dd, J = 8.2, 6.0 Hz), 3.48 (ddd, J = 9.7, 9.7, 4.5 Hz),
and 3.27 (dd, J = 11.3, 4.0 Hz), indicative of secondary alcoholic
functions, along with signals at δ 4.14 and 4.07 (each, br s) corre-
"
"
sponding to a primary alcoholic function (l Table 2). On the basis
epoxycycloartane (l Fig. 1).
ofDQF-COSY, HSQC, andHMBC spectra andbycomparison of these
data with those of cycloastragenol [22], it was observed that the
aglycone of compound 1 differed from cycloastragenol only in the
presence of a –COCH2OH group. The HMBC correlation between
the proton signal at δ 5.49 (H-16) and the carbon resonance at δ
175.7 (−COCH2OH) confirmed thelocation of the -COCH2OH group
at C-16. Thus the aglycone of 1 was identified as 16-O-hydroxyace-
toxy-3β,6α,16β,25-tetrahydroxy-20(R),24(S)-epoxycycloartane,
and, consequently, the structure of compound 1 was established
as 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl]-16-
O-hydroxyacetoxy-3β,6α,16β,25-tetrahydroxy-20(R),24(S)-epox-
The ESIMS spectrum of 5 showed the [M + Na]+ ion at m/z 791.
The MS/MS of this ion showed peaks at m/z 645 [M + Na-146]+,
due to the loss of a deoxyhexose unit, at 495 [M + Na-146-150]
+, corresponding to the loss of a xylopyranosyl unit.
A detailed analysis of the NMR data (1H, 13C, HSQC, HMBC, COSY)
of compound 5 in comparison with those of 4 showed the same
aglycone moiety in the two compounds.
Moreover, the 1H NMR spectrum of 5 showed two anomeric pro-
tondoublets atδ 4.78 (J = 1.2 Hz) and 4.28 (J = 7.5 Hz) in the down-
"
field region (l Table 1). The determination of the sequence and
linkage sites was obtained from the HMBC correlations between
the proton signals at δ 4.78 (H-1rha) and the carbon resonance at δ
81.3 (C-6), and between the proton signal at δ 4.28 (H-1xyl) and the
carbon resonance at δ 89.7 (C-3). Thus, compound 5 was identified
as 3-O-β-D-xylopyranosyl-6-O-α-L-rhamnopyranosyl-3β,6α,16β,
"
ycycloartane (l Fig. 1).
The 1H and 13C NMR spectroscopic data of the aglycone portion of
2 were similar to those of 1 except for the presence of signals due
to an additional secondary alcoholic function (δ 4.47, ddd, J = 8.8,
"
"
6.0, 3.5 Hz; δC 74.5) (l Table 2). In the HMBC spectrum, cross-
25-tetrahydroxy-20(R),24(R)-epoxycycloartane (l Fig. 1).
peaks between the proton signals at δ 4.47, 1.19 (Me-26), and
1.29 (Me-27) with the carbon at δ 72.6 (C-25) suggested the
placement of this additional secondary alcoholic function at C-
23. The α-orientation of the hydroxyl group at C-23 was deduced
from the J values of the signal corresponding to H-23 (8.8, 6.0,
3.5) in comparison with literature values [23]. Therefore, the
structure of compound 2 was established as 3-O-[α-L-rhamno-
pyranosyl-(1 → 2)-β-D-glucopyranosyl]-16-O-hydroxyacetoxy-
3β,6α,16β,23α,25-pentahydroxy-20(R),24(S)-epoxycycloartane
The positive ESIMS spectrum of 6 showed the [M + Na]+ ion at m/z
659. Its MS/MS fragmentation showed a peak at m/z 513 [M + Na-
146]+ due to the loss of a deoxyhexose unit. A detailed analysis of
the NMR data (1H, 13C, HSQC, HMBC, COSY) of compound 6 re-
vealed that while the aglycone moiety was the same as in 4, the
"
sugar portion corresponded to an α-rhamnopyranose unit (l Ta-
ble 1). In the HMBC spectrum a key correlation peak was ob-
served between the anomeric proton signal at δ 4.80 and the car-
bon resonance at δ 81.2 (C-6). Therefore, the structure of 6 was
established as 6-O-α-L-rhamnopyranosyl-3β,6α,16β,25-tetrahy-
"
(l Fig. 1).
"
The NMR data of the aglycone portion of 3 differed from those of
cycloastragenol [22] only in the absence of the signal due to a sec-
ondary alcoholic function and the occurrence of a typical reso-
droxy-20(R),24(R)-epoxycycloartane (l Fig. 1).
A detailed comparison of NMR (1H, 13C, HSQC, HMBC, COSY) and
ESIMS data of compounds 7–8 showed that the sugar chain was
identical in the two compounds. In particular, for the sugar por-
tion, compound 7 showed signals corresponding to three ano-
meric protons at δ 5.23 (d, J = 1.2 Hz), 4.82 (d, J = 7.5 Hz), and
nance of a keto group (δ 222.2) in the 13C NMR spectrum (l Table
"
2). The carbon resonances of the D ring and Me-18 in 3 suggested
that the keto group was located at C-16. This hypothesis was con-
firmed by the HMBC experiment, which showed diagnostic long-
range correlations between the proton signal at δ 1.28 (Me-18)
and the carbon resonance atδ 66.9 (C-17), and between the proton
signals at δ 2.98 (H-17) and 2.05 (H2-15) with the carbon reso-
nance at δ 222.2. From this evidence, in combination with the data
of the sugar moiety reported above, the structure of 3 was estab-
lished as 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyrano-
syl]-3β,6α,25-trihydroxy-20(R),24(S)-epoxycycloartane-16-one
"
4.44 (d, J = 7.5 Hz) (l Table 3). On the basis of 2D NMR data, one
α-rhamnopyranosyl (δ 5.23), one β-xylopyranosyl (δ 4.82), and
one β-glucuronic acid (δ 4.44) were identified. The determination
of the sequence and linkage sites was obtained from the HMBC
correlations between the proton signal at δ 5.23 (H-1rha) and the
carbon resonance at δ 79.3 (C-2xyl), the proton signal at δ 4.82 (H-
1
xyl) and the carbon resonance at δ 78.8 (C-2glcA), and the proton
signal at δ 4.44 (H-1glcA) and the carbon resonance at δ 92.4 (C-3).
Thus, the sugar sequence of compounds 7–8 was established as
3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 2)-β-
D-glucuronopyranoside. Moreover, the aglycone of compounds
7–10 was recognized as an oleanane-type triterpene by 1H NMR
"
(l Fig. 1).
The full assignments of the proton and carbon signals of the agly-
cone moiety of 4, secured by DQF-COSY, HSQC, and HMBC spec-
tra, were in good agreement with those of cycloastragenol [22],
except for the chemical shift of C-24 (δ 88.5), suggesting a change
of the absolute configuration at this center. The R configurations
and 13C NMR analyses (l Table 4) [25,26].
"
Gülcemal D et al. Triterpene Glycosides from… Planta Med 2012; 78: 720–729

728 Original Papers
The ESIMS spectrum of 7 showed the [M + Na]+ ion peak at m/z
967. The MS/MS spectrum of this ion showed peaks at m/z 821
[M + Na-146]+, due to the loss of a deoxyhexose unit and at m/z
689 [M + Na-146-132]+, corresponding to the loss of a xylopyra-
nosyl unit. The 1H NMR spectrum of 7 showed signals for six
methyl groups at δ 1.24, 1.23, 1.03, 1.00, 0.98, 0.90, one olefinic
proton at δ 5.27 (H-12, d, J = 3.5 Hz), three oxygen-bearing meth-
ine protons at δ 3.35 (H-3, dd J = 11.5, 4.5 Hz), 3.83 (H-21, brs),
3.36 (H-22, dd J = 3.5) and signals for two primary alcoholic func-
tions at δ 3.23 and 4.12 (H2-24, d J = 11.5), and 3.12 and 3.45 (H2-
as 29-O-β-D-glucopyranosyl-3β,22β,24,29-tetrahydroxy-olean-
12-ene (l Fig. 2).
"
Additionally, three known cycloartane-type glycosides, 25-O-
glucopyranosylcycloastragenol (11) [29], cycloaraloside D (12)
[31], and 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyrano-
syl]-25-O-β-D-glucopyranosyl-20(R),24(S)-epoxy-3β,6α,16β,25-
tetrahydroxycycloartane (13) [11], and two known oleanane gly-
cosides, astrajanoside A (14) [4] and astragaloside VIII (15) [29],
were isolated.
On the basis of the cytotoxic activities exhibited by cycloartane-
and oleanane-type glycosides isolated from Astragalus species
[15,19] and on the basis of cancer chemopreventive effects re-
ported for the natural and semisynthetic cycloartane-type and
related triterpenoids [32], the antiproliferative activity of com-
pounds 1–15 was tested in different cancer cell lines including
Hela (human cervical cancer cells), H-446 (human lung cancer
cells), HT-29 (human colon carcinoma cells), and U-937 (human
leukemia cells). In a range of concentrations between 1 and
50 µM, none of the tested compounds, except compound 8,
caused a significative reduction of the cell number when com-
pared with controls. Compound 8 displayed weak cytotoxicity
against HeLa and HT-29 cell lines with IC50 values of 36 and
50 µM, respectively. Etoposide, used as a positive control, showed
IC50 values ranging from 2 µM (U-937 cells) to 12 µM (H-446
cells).
"
29, d, J = 10.5) (l Table 4). Detailed NMR studies allowed us to
identify the aglycone of 7 as kudzusapogenol A [27]. The β-orien-
tation of the hydroxyl group at C-21 and the α-orientation of the
hydroxyl group at C-22 were derived from the ROESY spectrum,
which showed key correlation peaks between H-21 (δ 3.83) and
the proton signals H-29 (3.45) and H-19α (2.12), and between H-
22 (δ 3.36) and Me-28 (0.98). Thus, compound 7 was identified as
the new 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-
(1 → 2)-β-D-glucuronopyranosyl]-3β,21β,22α,24,29-pentahy-
"
droxyolean-12-ene (l Fig. 2).
The 1H NMR spectrum of 8 displayed signals for six tertiary methyl
groups at δ 1.24 (6H), 1.18, 1.00, 0.91, 0.83, for an olefinic proton
at δ 5.31 (t, J = 3.5 Hz), two oxygen-bearing methine protons at δ
3.36 (H-3, dd, J = 11.5, 4.5 Hz) and 3.52 (H-22, brs), and one pri-
mary alcoholic function at δ 4.11 and 3.23 (H2-24, d, J = 11.5 Hz)
"
(l Table 4). A combination of 1D and 2D NMR techniques led to
1
the assignment of H and 13C NMR signals due to A, B, C, and D
rings of a 3β,24-dihydroxyolean-12-ene sapogenol unit [28], gly-
cosylated at C-3. On the basis of NMR data, the E ring exhibited an
oxymethine at C-22 (δC 77.3, δH 3.52 brs) and a -COOH group at
C-30 (δC 187.8). Thus, the structure of compound 8 was eluci-
dated as 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-
(1 → 2)-β-D-glucuronopyranosyl]-3β,22β,24-trihydroxyolean-12-
Acknowledgements
!
The authors are grateful to Nihat Şahin and Dr. Serdar G. Şenol for
the collection and identification of the plant material.
"
en-29-oic acid (l Fig. 2).
Conflict of Interest
!
The 1H NMR spectrum of 9 displayed signals for seven tertiary
methyl groups at δ 1.25, 1.16, 1.06, 1.00, 0.94, 0.93, and 0.90,
The authors declare that there is no conflict of interest.
"
and for an olefinic proton at δ 5.31 (1H, t, J = 3.5 Hz) (l Table 4).
On the basis of NMR data in comparison with those of astragalo-
side VIII [29], it clearly appeared that compound 9 differed from
astragaloside VIII only in the presence of an additional α-arabino-
pyranosyl unit (H-1ara δ 4.27, d, J = 3.7 Hz), placed at position C-
22, on the basis of the HMBC correlation between the proton sig-
nal at δ 4.27 (H-1ara) and the carbon resonance at δ 83.3 (C-22).
Therefore, the structure of 9 was established as 3-O-[α-L-rhamno-
pyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 2)-β-D-glucurono-
pyranosyl]-22-O-α-L-arabinopyranosyl-3β,22β,24-trihydroxyolean-
References
1 Podlech D. Phylogeny and progression of characters in Old World Astra-
gali (Leguminosae). In: Zhang A, Wu S, editors. Floristic characteristics
and diversity of East Asian plants. Beijing, China: China Higher Educa-
tion Press; 1998: 405–407
2 Davis PH. Flora of Turkey and the East Aegean Islands, Volume 3. Edin-
burgh: Edinburgh University Press; 1970: 49–254
3 Bedir E, Calış I, Aquino, R, Piacente S, Pizza C. Cycloartane triterpene gly-
cosides from the roots of Astragalus brachypterus and Astragalus mi-
crocephalus. J Nat Prod 1998; 61: 1469–1472
4 Bedir E, Calış I, Aquino R, Piacente S, Pizza C. Secondary metabolites
from the roots of Astragalus trojanus. J Nat Prod 1999; 62: 563–568
5 Bedir E, Calış İ, Aquino R, Piacente S, Pizza C. Trojanoside H: a cycloar-
tane-type glycoside from the aerial parts of Astragalus trojanus. Phyto-
chemistry 1999; 51: 1017–1020
6 Bedir E, Çalış I, Dunbar C, Shara, R, Buolamwini JK, Khan IA. Two novel
cycloartane-type triterpene glycosides from the roots of Astragalus
prusianus. Tetrahedron 2001; 57: 5961–5966
7 Calış İ, Yuruker A, Taşdemir D, Wrigh AD, Sticher O, Luo YD, Pezzuto JM.
Cycloartane triterpene glycosides from the roots of Astragalus melano-
phrurius. Planta Med 1997; 63: 183–186
8 Gulcemal D, Alankuş-Calışkan O, Perrone A, Ozgokçe F, Piacente S, Bedir
E. Cycloartane glycosides from Astragalus aureus. Phytochemistry
2011; 72: 761–768
9 Yalcin FN, Placente S, Perrone A, Capasso A, Duman H, Calis I. Cycloar-
tane glycosides from Astragalus stereocalyx. Phytochemistry 2012; 73:
119–126
"
12-ene (l Fig. 2).
The ESIMS spectrum of 10 showed the [M + Na]+ ion at m/z 659.
The MS/MS of this ion showed a peak at m/z 439 [M + Na-180]+
due to the loss of a hexose unit. A combination of 1D and 2D NMR
techniques led to the assignment of 1H and 13C NMR signals due
to the A, B, C, D rings of a 3β,24-dihydroxyolean-12-ene sapoge-
nol unit [30]. The E ring was shown to contain an oxymethine at
C-22 (δC 77.1, δH 3.50 brs) and a glycosylated hydroxymethyl
group at C-29 (δC 80.2, δH 3.17 and 3.68, each d, J = 9.7 Hz). The
occurrence of a 29-CH2OR function was derived from the reso-
nance of Me-30 at δ 24.4, superimposable to that of abrisapoge-
nol B [30]. 1D and 2D NMR data also indicated the presence of a
terminal β-D-glucopyranosyl moiety, which was located at C-29
on the basis of the HMBC correlation between H-1glc (δ 4.26)
and C-29 (δ 80.2) signals. Thus, the structure of 10 was elucidated
10 Horo I, Bedir E, Perrone A, Ozgokçe F, Piacente S, Alankuş-Calışkan O. Tri-
terpene glycosides from Astragalus icmadophilus. Phytochemistry
2010; 71: 956–973
Gülcemal D et al. Triterpene Glycosides from… Planta Med 2012; 78: 720–729

Original Papers 729
11 Polat E, Bedir E, Perrone A, Piacente S, Alankus-Calıskan O. Triterpenoid
saponins from Astragalus wiedemannianus Fischer. Phytochemistry
2010; 71: 658–662
12 Bedir E, Pugh N, Calış I, Pasco DS, Khan IA. Immunostimulatory effects of
cycloartane-type triterpene glycosides from Astragalus species. Biol
Pharm Bull 2000; 23: 834–837
13 Ozipek M, Dönmez AA, Calış I, Brun R, Ruedi P, Taşdemir D. Leishmani-
cidal cycloartane-type triterpene glycosides from Astragalus oleifolius.
Phytochemistry 2005; 66: 1168–1173
14 Gariboldi P, Pelizzoni F, Tatò M, Verotta L, El-Sebakhy NA, Asaad AM,
Abdallah RM, Toaima SM. Cycloartane triterpene glycosides from Astra-
galus trigonus. Phytochemistry 1995; 40: 1755–1760
15 Radwan MM, El-Sebakhy NA, Asaad AM, Toaima SM, Kingston DGI.
Kahiricosides II–V cycloartane glycosides from an Egyptian collection
of Astragalus kahiricus. Phytochemistry 2004; 65: 2909–2913
16 Khushbaktova ZA, Agzamova MA, Syrov VN, Radchenko NV, Mirsalikhova
NM, Umarova FT. Influence of cycloartanes from plants of the genus
Astragalus and their synthetic analogs on the contractive function of
the myocardium and the activity of Na,K-ATPase. Chem Nat Compd
1994; 30: 469–473
17 Sevimli-Gur C, Onbaşılar İ, Atilla P, Genç R, Cakar N, Deliloğlu-Gurhan I,
Bedir E. In vitro growth stimulatory and in vivo wound healing studies
on cycloartane-type saponins of Astragalus genus. J Ethnopharmacol
2011; 134: 844–850
22 Kitagawa I, Wang HK, Saito M, Takagi A, Yoshikawa M. Saponin and
sapogenol. XXXV. Chemical constituents of Astragali radix, the root
of Astragalus membranaceus Bunge. (2). Astragalosides I, II and IV,
Acetylastragaloside I and isoastragalosides I and II. Chem Pharm Bull
1983; 31: 698–708
23 Calış I, Donmez AA, Perrone A, Pizza C, Piacente S. Cycloartane saponins
from Astragalus campylosema Boiss. ssp. campylosema. Phytochemistry
2008; 69: 2634–2638
24 Mamedova RP, Isaev MI. Triterpenoids from Astragalus plants. Chem
Nat Compd 2004; 40: 303–357
25 De Tommasi N, Piacente S, Gacs-Baitz E, De Simone F, Pizza C, Aquino R.
Triterpenoid saponins from Spergularia ramosa. J Nat Prod 1998; 61:
323–327
26 Kapusta I, Stochmal A, Perrone A, Piacente S, Pizza C, Oleszek W. Triter-
pene saponins from Barrel Medic (Medicago truncatula) aerial parts.
J Agric Food Chem 2005; 63: 2164–2170
27 Kinjo J, Miyamoto I, Murakami K, Kida K, Tomimatsu T, Yamasaki M,
Nohara T. Oleanene-sapogenols from puerariae radix. Chem Pharm
Bull 1985; 33: 1293–1296
28 Kitagawa, I, Wang HK, Saito M, Yoshikawa M. Saponin and sapogenol.
XXXIII. Chemical constituents of the seeds of Vigna angularis (WILLD.)
OHWI et OHASHI. (3). Azukisaponins V and VI. Chem Pharm Bull 1983;
31: 683–688
29 Kitagawa I, Wang HK, Yoshikawa M. Saponin and sapogenol. XXXVII.
Chemical constituents of Astragali radix, the root of Astragalus mem-
branaceus Bunge. (4). Astragalosides VII and VIII. Chem Pharm Bull
1983; 31: 716–722
18 Nalbantsoy A, Nesil T, Erden S, Calış I, Bedir E. Adjuvant effects of Astra-
galus saponins macrophyllosaponin B and astragaloside VII. J Ethno-
pharmacol 2011; 134: 897–903
19 Kikuchi T, Akihisa T, Tokuda H, Ukiya M, Watanabe K, Nishino H. Cancer
chemopreventive effects of cycloartane-type and related triterpenoids
in in vitro and in vivo models. J Nat Prod 2007; 70: 918–922
20 De Marino S, Borbone N, Iorizzi M, Esposito G, McClintock JB, Zollo F. Bio-
active asterosaponins from the starfish Luidia quinaria and Psilaster
cassiope. Isolation and structure characterization by two-dimensional
NMR spectroscopy. J Nat Prod 2003; 66: 515–519
30 Miyao H, Sakai Y, Takashita T, Kinjo J, Nohara T. Triterpene saponins
from Abrus cantoniensis (Leguminosae). I. Isolation and characteriza-
tion of four new saponins and a new sapogenol. Chem Pharm Bull
1996; 44: 1222–1227
31 Isaev MI. Triterpene glycosides of Astragalus and their genins XXXIX.
Cycloaraloside D from Astragalus amarus. Chem Nat Compd 1991; 4:
526–528
21 Mosmann T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J Immunol Meth-
ods 1983; 65: 55–63
32 Tian Z, Yang M, Huang F, Li K, Si J, Shi L, Chen S, Xiao P. Cytotoxicity of
three cycloartane triterpenoids from Cimicifuga dahurica. Cancer Lett
2005; 226: 65–75
Gülcemal D et al. Triterpene Glycosides from… Planta Med 2012; 78: 720–729