Three new cycloartane triterpenoids from Astragalus bicuspis

Article abstract of DOI:10.1055/s-0030-1271196

Three new cycloartane triterpenoids have been isolated from Astragalus bicuspis Fisch. Their structures were elucidated as 23(R),24(S),25(R),26(S)-16/ 23,23/26,24/25-triepoxy-26-hydroxy-9,19-cyclolanosta-3,6-dione (1), 6,23,24,25-tetraol-16-acetoxy-23(R),24(R)-9,19-cyclanosta-3-one (2), and 6,23,24,25-tetraol-16-acetoxy-23(R),24(R)-9,19-cyclolanosta-3-O-xyloside (3), based on their spectroscopic analysis. All cycloartane tritepenoids exhibited weak cytotoxicities against 3T3 fibroblast cells as compared to the standard drug cycloheximide. Compounds 3 and 4 were also tested for their antileishmanial potential, and a weak activity was observed against promastigotes of Leishmania major. Georg Thieme Verlag KG Stuttgart · New York.

Full text of DOI:10.1055/s-0030-1271196

Original Papers 1829
Three New Cycloartane Triterpenoids
from Astragalus bicuspis
Saleem Jan1,3, Ahmed Abbaskhan , Syed Ghulam Musharraf , Samina A. Sattar , Samreen ,
1
1
1
1
Authors
2
2
2
1
1,2
Saud I. Resayes , Zeid A. Al-Othman , Abdullah M. Al-Majid , Atta-ur-Rahman , M. Iqbal Choudhary
1
Affiliations
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi, Pakistan
Department of Chemistry, King Saud University, Riyadh, Saudi Arabia
Department of Chemistry, University of Science and Technology, Bannu, Pakistan
2
3
Key words
Abstract
23(R),24(R)-9,19-cyclolanosta-3β-O-xyloside (3),
"
l Astragalus bicuspis
!
based on their spectroscopic analysis. All cycloar-
tane tritepenoids exhibited weak cytotoxicities
against 3T3 fibroblast cells as compared to the
standard drug cycloheximide. Compounds 3 and
4 were also tested for their antileishmanial poten-
tial, and a weak activity was observed against
promastigotes of Leishmania major.
"
l Leguminosae
Three new cycloartane triterpenoids have been
isolated from Astragalus bicuspis Fisch. Their
structures were elucidated as 23(R),24(S),25(R),
26(S)-16β/23,23/26,24/25-triepoxy-26-hydroxy-
"
l cycloartane triterpenoids
"
l antileishmanial activity
"
l cytotoxicity
"
l 3T3 fibroblast cell line
9,19-cyclolanosta-3,6-dione (1), 6α,23,24,25-tet-
raol-16β-acetoxy-23(R),24(R)-9,19-cyclanosta-3-
one (2), and 6α,23,24,25-tetraol-16β-acetoxy-
Introduction
!
Materials and Methods
!
Plants of the genus Astragalus Linn. (Legumino-
sae) are used in traditional medicines such as
antiperspirants, diuretics, and as a tonic [1]. Sev-
eral species are used for the treatment of nephri-
tis, diabetes, leukemia, and uterine cancer [2].
Some of these plants contain cytotoxic constitu-
ents while some other species stimulate lympho-
cyte transfer in vitro [3]. These plants also pro-
mote the discharge of pus and the growth of new
tissues [4]. The chemical constituents of these
plants exhibited antitumor, immunodepressant,
and antiviral activities [5,6].
General experimental procedures
IR spectra were recorded as KBr discs on a JASCO
A-302 spectrophotometer. Optical rotations were
measured on a digital JASCO DIP-360 polarimeter
1
in MeOH. The H NMR spectra were recorded on a
Bruker AV-300 and AVX-600 spectrometers, oper-
1
3
ating at 300 and 600 MHz, respectively. C NMR
spectra were taken on Bruker AV-300 and AV-
600 spectrometers operating at 75 and 150 MHz,
respectively. The HMQC (heteronuclear multiple
quantum coherence) and HMBC (heteronuclear
multiple bond connectivity) spectra were mea-
sured on a Bruker AV-400 and AV-600 spectrom-
eters. The chemical shifts were reported in δ
(ppm), referenced with respect to the residual
solvent signals of C H N/MeOH/CHCl . Coupling
received
revised
accepted
March 10, 2011
May 10, 2011
May 13, 2011
Bibliography
In previous phytochemical studies, we have iso-
lated five cycloartane triterpenoids [7]. In contin-
uation of these studies, we have further isolated
DOI http://dx.doi.org/
0.1055/s-0030-1271196
1
Published online July 15, 2011
Planta Med 2011; 77:
"
four cycloartane triterpenoids (see l Fig. 1) from
5
5
3
1
829–1834 © Georg Thieme
the methanolic extract of A. bicuspis. Compounds
constants (J) were measured in Hz. Mass spectra
were recorded on a Q-STAR XL (Applied Biosys-
tems). Each compound (4 µg/mL), dissolved in
acetonitrile: 0.1% HCOOHaq (2:1), was infused in-
to the mass spectrometer at a flow rate of 3 µL/
min to acquire full scan and product ion mass
spectra. The electrospray voltage at the spraying
needle was optimized at 5200 V for positive
modes of ionization. Low-energy CID (collision-
induced dissociation) experiments were per-
formed by using nitrogen (CID gas valve set to 4)
as a collision gas, and collision energy of 10–40 eV
was used. FAB‑MS were measured on a Jeol HX
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
3
and 4 showed weak antileishmanial activity
against Leishmania major promastigotes as com-
pared to the standard drugs pentamidine and
amphotericin B. Compounds 1–4 exhibited weak
to moderate cytotoxicities against the 3T3 fibro-
blast cell (mouse) as compared to standard drug
cycloheximide.
Correspondence
Prof. M. Iqbal Choudhary
H.E.J. Research Institute
of Chemistry
International Center for
Chemical and Biological
Sciences
University of Karachi
Karachi 75270
Pakistan
Phone: + 922148249245
Fax: + 922148190189
hej@cyber.net.pk
Jan S et al. Three New Cycloartane… Planta Med 2011; 77: 1829–1834

1
830 Original Papers
Fig. 1 Structures of the compounds 1–5 and
bicuspiside C.
110 mass spectrometer. Polyamide-6 DF (Riedel De Haen AG) and
compounds 1 (6 mg) and 2 (5 mg) with 2% MeOH: CHCl elution.
3
silica gel (E. Merck; 160–200 µM mesh) were used as stationary
phases in column chromatography.
Another fraction, obtained at 30% MeOH: CHCl3 elution (40 g),
was subjected to silica gel and polyamide column chromatogra-
phy to yield 9 small fractions. One of these fractions (9 g), eluted
Plant material
from polyamide column at 3% MeOH: CHCl , upon further silica
3
The whole plant of A. bicuspis Fisch. was collected from Khal-
gel CC, afforded compounds 3 (11 mg) and 4 (4 mg) at 3% MeOH:
taran-Haramosh, Gilgit valley (Pakistan) in the flowering season
CHCl and 2% MeOH: CHCl elutions, respectively. The same frac-
3
3
(
July 2003). The plant was identified by Sher Wali Khan, a taxon-
tion (9 g), eluted from polyamide column at 3% MeOH: CHCl3,
omist of the Department of Botany, University of Karachi, Paki-
stan. The voucher specimen (# 67854) was deposited at the her-
barium of the same department.
upon further silica gel CC, yielded compound 5 (6 mg) at 2%
MeOH: CHCl elution.
Bicusposide D (1): amorphous powder; [α]
3
25
D
− 11.0 (c 0.5, MeOH);
IR (KBr): νmax = 3464 (-OH), 1708 (ketone carbonyl), 2933 (CH -
2
−
1
+
Extraction and isolation
cyclopropane) cm ; ESI-QTOF‑MS: m/z = 499.3061 [M + H]
(calcd. for C30H43O : 499.3060); FAB‑MS (−ve): m/z = 497; 1H-
Air-dried plant material (2 kg) was extracted with 80% aqueous
MeOH under reflux (5 L × 3) for five hours. The extract was fil-
tered and evaporated to dryness. The methanolic extract (120 g)
was subjected to vacuum liquid chromatography (VLC) by using
silica gel (1000 g, 160–200 µM). The elution was carried out in a
6
1
3
"
and C‑NMR (pyridine-d ) spectroscopic data: see l Table 1.
Bicusposide E (2): amorphous powder; [α]
5
25
D
− 17.0 (c 0.5, MeOH);
IR (KBr): νmax = 3424 (-OH), 1721, 1728 (ketone and ester carbon-
−
1
yls), 2935 (CH -cyclopropane) cm ; ESI-QTOF‑MS: m/z =
2
549.3798 [M + H] (calcd. for C32H53O : 549.3791); 1H- and
+
gradient manner by using 20% CHCl : n-hexane (5 L × 3 times),
3
7
1
3
"
3
0% CHCl : n-hexane (5 L × 3 times), 50% CHCl : n-hexane (5 L ×
C‑NMR (pyridine-d ) spectroscopic data: see l Table 1.
5
3
3
25
2
times), 70% CHCl : n-hexane (5 L × 3 times), CHCl (5 L × 3
D
Bicusposide F (3): amorphous powder; [α] − 3.7 (c 0.5, MeOH), IR
3
3
times), 30% MeOH: CHCl3 (10 L × 3 times), 50% MeOH: CHCl3
10 L × 2 times), and 100% MeOH (15 L). Eight main fractions (1–
) were obtained.
(KBr): ν
= 3408 (-OH), 1728 (ester), 2928 (CH -cyclopropane)
max
2
−1
+
(
8
cm ; ESI-QTOF‑MS: m/z = 683.4336 [M
C37H63O11: 683.4370); FAB‑MS (+ve): m/z = 683; FAB‑MS (−ve):
+
H] (calcd. for
1
13
One of the above fractions (3 g), obtained at 20% CHCl : n-hexane
m/z = 681; H- and C‑NMR (pyridine-d ) spectroscopic data:
3
5
"
elution, was subjected to further silica gel CC which afforded
see l Table 1.
Jan S et al. Three New Cycloartane… Planta Med 2011; 77: 1829–1834

Original Papers 1831
Table 1 1H- and 13C-NMR data of compounds 1–3 in pyridine-d5 (δ in ppm, J in Hz).
Position
1
2
3
δΗ (mult.)
δC
δΗ (mult.)
δC
δΗ (mult.)
δC
1
2
3
4
5
6
7
8
9
1.62 m, 1.21 m
30.5
35.9
214.0
49.9
55.4
210.6
41.5
41.7
22.2
28.6
26.6
32.9
47.1
45.0
41.2
74.0
56.3
18.4
20.3
25.9
20.9
42.3
105.8
64.0
63.9
97.7
13.1
25.5
17.5
20.9
–
1.59 m,1.25 m
31.8
35.7
216.8
50.5
53.5
69.0
38.0
48.0
21.3
28.2
26.0
32.6
46.8
46.0
46.1
74.5
56.2
19.1
30.7
26.6
17.8
42.4
70.8
79.3
74.6
29.3
24.1
28.5
20.3*
20.2*
21.6
170.9
–
1.62 m,1.23 m
32.5
30.4
88.7
42.7
54.1
67.7
38.2
46.7
21.2
29.2
26.2
32.8
47.0
46.2
45.8
74.7
56.2
18.5
29.9
26.7
17.9
42.5
70.8
79.4
74.6
29.3
24.2
28.8
16.7
20.0
21.6
170.8
107.6
75.7
78.6
71.3
67.1
a
a
a
a
a
a
2.60 , 2.05
2.72 , 2.43
2.60 , 2.28
–
–
3.62 dd (11.7, 4.8)
–
–
–
2.83 s
1.75a
3.76a
1.75 d (9.3)
3.76a
–
a
a
2.60 , 2.25
1.70 m, 1.54 m
1.72a
1.79 m, 1.62 m
1.89a
1.83 dd (2.4, 5.1)
–
–
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
–
–
–
1.88 m,1.20 m
1.50 m,1.21 m
–
1.99 m, 0.99 m
1.91 m, 1.28 m
–
1.76 m, 1.25 m
1.64 m, 1.28 m
–
–
–
–
1.82 m, 1.56 m
4.57 q (7.7)
1.65 m
2.19 m, 1.35 m
5.66 ddd (7.9, 7.8, 4.7)
1.85 m
2.19 m, 1.35 m
5.68 ddd (7.8, 7.5, 4.5)
1.90 m
1.08 s
1.19 s
1.20 s
0.33, 0.22 each d (5.1)
1.88 m
0.66, 0.33 each d (3.5)
1.87 m
0.56, 0.26 each d (4.1)
1.92 m
0.94 d (5.5)
1.11 d (6.4)
1.11 d (6.4)
a
a
a
a
a
a
2.19 , 1.90
2.20 , 2.00
2.40 , 2.00
–
4.43 br t (9.2)
4.42 br t (8.4)
3.84 s
3.70 d (8.6)
3.71 d (8.5)
–
–
–
5.82 s
1.72 s
1.66 s
1.75 s
1.46 s
0.88 s
2.19 s
–
1.66 s
1.73 s
1.71 s
1.56 s
1.98 s
1.24 s
1.34 s
0.78 s
0.95 s
CH3CO
–
–
–
–
–
–
–
2.19 s
CH3CO
–
–
1
2
3
4
5
–
–
4.90 d (7.4)
4.06 br t
–
–
–
–
–
–
4.15 t (8.5)
4.21a
4.37 dd (10.8, 4.8), 3.69a
–
–
–
–
–
–
a
Overlapped signals; * assignments can be exchanged; C corresponding carbons with 13C-NMR data
Acid hydrolysis of compound 3
Compound 3 (0.9 mg) was refluxed with 6% HCl-MeOH-n-BuOH
manial activity were calculated by Ezfit 5.03 (Perrella Scientific
Software). Pentamidine (IC50 = 4.32 ± 0.09 µM, purchased from
ICN, Biomedical Inc.) and amphotericin B (IC50 = 0.54 ± 0.02 µM,
purchased from ICN, Biomedical, Inc.) were used as positive con-
trols.
(5 mL, 2:1:1 v/v/v) for 1 h at 90°C. The mixture was neutralized
with 1 M KOH, and the presence of xylose sugar was deduced by
1
H NMR (CD OD) spectroscopy as well as through TLC compari-
3
son with standard xylose.
Cytotoxicity
In vitro leishmanicidal activity
Cytotoxicity of compounds was evaluated in 96-well flat-bottom
microplates using the standard MTT (3-[4,5-dimethylthiazole-2-
yl]-2,5-diphenyl-tetrazolium bromide) colorimetric assay [21,
22]. 3T3 Cells (mouse fibroblasts) were cultured in Dulbeccoʼs
modified Eagleʼs medium, supplemented with 5% of fetal bovine
serum (FBS), 100 IU/mL of penicillin, and 100 µg/mL of strepto-
Leishmania major (DESTO) promastigotes were grown at 22–25°C
in RPMI-1640 medium [20] (Sigma) containing 10% heat-inacti-
vated fetal bovine serum (FBS). A logarithmic phase of growth
was maintained. The final concentration of parasites was ad-
justed to 1 × 106 cells/mL. The test compound (1 mg) was dis-
solved in 50 µL DMSO, and the volume was adjusted to 1 mL with
complete media. In a 96-well microtiter plate, 180 µL of medium
was added in different wells. Then 20 µL of the test compound
was added in the medium and diluted. A total of 100 µL of para-
site suspension was added into each well of the 96-well plates.
Plates were incubated at 21–22°C for 72 hrs, and cultures were
examined microscopically on an improved Neubauer counting
chamber. The IC50 values of compounds possessing antileish-
2
mycin by using 25 cm flasks in a 5% CO2 incubator at 37°C.
Growing cells were harvested, counted with hemocytometer
and diluted with a particular medium. Cell cultures with the con-
4
centration of 3 × 10 cells/mL were prepared and placed (100 µL/
well) onto 96-well plates. After overnight incubation, medium
was removed, and 200 µL fresh medium was added with different
concentrations of compounds (1–100 µM). 50 µL MTT (2 mg/mL)
was added after 72 hrs to each well, and incubation was contin-
Jan S et al. Three New Cycloartane… Planta Med 2011; 77: 1829–1834

1
832 Original Papers
ued for 4 hrs. Subsequently, 100 µL of DMSO was added to each
well. The extent of MTT reduction to formazan within cells was
calculated by the measurement of the absorbance at 540 nm by
a microplate ELISA reader. The cytotoxicity was recorded as the
concentration causing 50% growth inhibition. Cycloheximide
(95% pure, purchased from Sigma-Aldrich Chemicals) was used
as a positive control.
Results and Discussion
!
The powdered plant material (whole plant) of A. bicuspis Fisch.
was extracted with 80% aqueous methanol. The MeOH extract
was fractionated by vacuum liquid chromatography by using sili-
ca gel. Crude fraction eluted with 30% MeOH: CHCl was further
3
subjected to silica gel and polyamide column chromatography to
obtain compounds 1–5.
ES-QTOF‑MS (electrospray quadrupole time-of-flight mass spec-
trometry) of 1 supported the formula C30H43O (calcd. 499.3060)
6
+
due to the [M + H] ion at m/z 499.3061. The molecular formula
was further supported by FAB‑MS (fast atom bombardment mass
spectrometry) showing a pseudomolecular ion (−ve mode) at m/z
−
4
97 [M − H] . An absorption band for cyclopropane of cycloartane
triterpene-type skeleton was present in the IR spectrum at
−
1
−1
2
933 cm along with the absorption of OH (3464 cm ) and ke-
−
1
tone functionalities (1708 cm ).
Fig. 2 Key dipolar couplings observed in the ROESY spectra of 1 and 2.
The compound 1 was found similar to bicusposide C with the ex-
ception of two ketonic functionalities in 1 instead of a β-xylose
and an α-hydroxy at C-3 and C-6, respectively, in bicusposide C
[
7]. A cycloartane-type skeleton was further inferred from the cy-
1.88 (H-20), and 5.82 (H-26) in the ROESY spectrum. Thus, the
relative configurations at C-23, C-24, C-25, and C-26 were deduced
as R, S, R, and S, respectively, by comparing them with β-oriented
CH -18 (δ 1.08) and H-20 (δH 1.88). The stereochemistry of CH3-
1
clopropyl proton signals in the H NMR spectrum at δ 0.22 and
0
1
Η
.33 (each 1H, d, J = 5.1 Hz) along with four tertiary methyls at δΗ
.08 (s, CH -18), 1.56 (s, CH -28), 1.24 (s, CH -29), and 0.78 (s,
3
3
3
3
H
CH -30). Additional methyl signals at δ 1.73 and 0.94 (3H, d,
18 and H-20 was deduced on the basis of biogenetic grounds in
cycloartanes. From these observations, the structure of com-
pound 1 was deduced as 23(R),24(S),25(R),26(S)-16β/23,23/
26,24/25-triepoxy-26-hydroxy-9,19-cyclolanosta-3,6-dione and
trivially named as bicusposide D.
3
Η
J = 5.5 Hz) were due to C-27 tertiary and C-21 secondary methyls,
respectively. The 13C NMR spectrum also contained resonances
for cyclopropyl carbons at δ 20.3 (CH ), 22.2 (C-9), and 28.6 (C-
C
2
1
0) [8] along with four tertiary methyl carbons at δ 18.4 (CH -
C 3
1
8), 25.5 (CH -28), 17.5 (CH -29), and 20.9 (CH -30). The carbon
The ESI‑MS (+ve) of 2 showed a peak at m/z 549.3798 [M + H]+
3
3
3
signals at δ 214.0 and 210.6 were due to two ketonic functional-
ities in the cycloartane skeleton. The presence of 16β/23,23/
(C32H53O , calcd. for 549.3791). The IR spectrum gave absorption
C
7
−
1
for cyclopropane of a cycloartane-type skeleton at 2935 cm ,
−
1
26,24/25-triepoxy moieties (H-16: δΗ 4.57; C-16: δC 74.0; C-23:
along with hydroxyl (3424 cm ) and ester carbonyl (1721 and
−
1
δC 105.8; H-26: δΗ 5.82; C-26: δC 97.7; C-23: δC 105.8; H-24: δΗ
1728 cm ) functionalities.
1
3
2
.84; C-24: δC 64.0; C-25: δC 63.9) and a hemiacetal moiety (H-
6: δΗ 5.82; C-26: δ 97.7; C-23: δ 105.8) was also deduced from
The H NMR spectrum of 2 also showed the main features of a cy-
cloartane-type triterpene. This includes cyclopropane methylene
C
"
C
the NMR data of 1 (l Table 1).
protons (δΗ 0.66, 0.33, J = 3.5 Hz, H -19), six tertiary methyls (δ
2
Η
HMBC (heteronuclear multiple bond correlation) data was very
0.88, 1.46, 1.66, 1.75, 1.72, 1.19 for CH -30, CH -29, CH -27,
3 3 3
useful for the structure elucidation of compound 1. H-5 (δ 2.83)
CH -28, CH -26, CH -18, respectively) and one secondary methyl
3 3 3
Η
showed 2JCH correlations both with C-4 (δC 49.9) and C-6 (δC
at δΗ 1.11 (d, J = 6.4 Hz, CH -21). Additionally, the resonance for
3
1
2
10.6) whereas a hemiacetal proton (H-26, δΗ 5.82) showed cor-
relations with the carbon signals at δC 105.8 (C-23) and 64.0 (C-
4) in the HMBC spectrum. Similarly, H-24 (δΗ 3.84) showed
HMBC interactions with carbon signals at δC 105.8 (C-23), 63.9
acetyl methyl protons at δ 2.19 also appeared in the H NMR
H
1
3
spectrum. The C NMR of compound 2 had 32 signals, two of
which were attributed to an acetyl group (δC 21.6 and 170.9),
while remaining signals were due to the triterpenoidal aglycone.
The NMR resonances of the aglycone moiety were in good agree-
ment with the cycloorbigenin-C [9], which is 23ξ,24ξ-cyclo-
2
(
C-25), and 13.1 (C-27). The ether connectivity between C-23 and
3
C-16 was determined based on the J correlation of H-16 (δΗ
CH
4
.57) with C-23 (δ 105.8).
artane-3β,6α,16β,23,24,25-hexaol, acylated at C-16 (O) [δ 74.7,
C-16, δΗ 5.66 ddd, J = 7.9, 7.8, 4.7 Hz, H-16]. The C‑NMR spec-
trum showed signals for three secondary carbinol C-atoms at δC
C
C
1
3
The relative configurations in 1 were inferred through ROESY ex-
periment (see l Fig. 2). The C-16 methine proton (δ 4.57)
"
Η
showed cross-peaks with the signals at δH 0.78 (H-30) and 1.65
69.0, 70.8, and 79.3 and for a tertiary carbinol at δ 74.6. The po-
C
(
H-17α) in the ROESY spectrum, indicating a β-orientation of the
sition of the acetoxy group at C-16 was unambiguously deduced
3
geminal 16-OH. The C-24 methine proton (δΗ 3.84) exhibited
cross-peaks with the signals at δΗ 1.73 (CH -27), 1.08 (CH -18),
by the J
correlations of H-16 (δΗ 5.68) with acetoxy carbonyl
C–H
carbon (δ 170.9) in the HMBC spectrum.
3
3
C
Jan S et al. Three New Cycloartane… Planta Med 2011; 77: 1829–1834

Original Papers 1833
The stereochemical assignments for C-23 and C-24 OH were non-
trivial due to free rotation of the side chain. However, based on
comparison with the literature data of similar triol- and side
chain-containing compounds, the stereochemistries at these cen-
ters of 2 were tertiary assigned [10,11]. The NOE and coupling
constants supported this assignment. The large coupling constant
of H-24 (J = 8.6 Hz) was an indication of a transoid arrangement
of diols [11]. Interestingly in the ROESY spectrum, the C-23 meth-
ine proton signal (δΗ 4.42 brt, J = 9.0 Hz) showed a cross-peak
with the C-20 methine proton. The orientation of H-20 was de-
duced on the basis of the reported biogenesis of cycloartanes.
Thus the stereochemistry of 23- and 24-OH was deduced as R,
by comparing it with reported data and on biogenetic grounds.
The α-orientation of 6-hydroxy and β-orientation of 16-acetoxy
On the basis of spectroscopic studies, the structure of compound
3 (bicusposide F) was deduced as 6α,23,24,25-tetraol-16β-ace-
toxy-23(R),24(R)-9,19-cyclolanosta-3β-O-xyloside.
The NMR data of 4 was found to be distinctly similar to cyclosi-
versigenin-3β-O-xyloside [7], except the presence of additional
signals for acetoxy functionality. It was identified as 20(R),24(S)-
epoxycycloartane-3β,6α,16β,25-tetraol-3β-O‑D-(2-O-acetyl)-xy-
lopyranoside, earlier reported from the Astragalus species [13].
Compound 5 was found to be an oleanane-type triterpene, based
on NMR data, and was identified as soyasapogenol B [14–19].
All cycloartane tritepenoids were tested for their cytotoxicity
against 3T3 fibroblast cells and compared with the standard drug,
cycloheximide (IC50 = 0.22 ± 0.12 µM). Weak cytotoxicities (IC50
=
> 100 µM, each) were exhibited by compounds 1–3 against the
3T3 fibroblast cells (mouse) whereas a moderate cytotoxicity
(IC50 = 49.33 ± 1.65 µM) was exhibited by 4.
was also deduced by ROESY cross-peaks. H-6 (δ 3.76, m) showed
Η
cross-peaks with H -19, while H-16 (δ 5.66, 1H, ddd, J = 7.9, 7.8,
2
Η
4
.7 Hz, H-16,) exhibited correlations with H-17 (δΗ 1.85, m) and
Compounds 3 and 4, obtained in good yield, were also tested for
their antileishmanial potential against Leishmania major pro-
mastigotes, by using pentamidine (IC50 = 4.32 ± 0.09 µM) and am-
photericin B (IC50 = 0.54 ± 0.02 µM) as standard drugs. Weak anti-
leishmanial activities with IC50 = 37.58 ± 0.60 µM and 121.22 ±
1.08 µM were exhibited by compounds 3 and 4, respectively.
CH -30 (δ 0.88, s).
On the basis of spectroscopic evidences, the structure of com-
pound 2 was deduced as 6α,23,24,25-tetraol-16β-acetoxy-23
3
Η
(
R),24(R)-9,19-cyclolanosta-3-one and was trivially named as bi-
cusposide E. The ESI‑MS (+ve) of 3 exhibited a peak at m/z
6
+
83.4336 [M + H] (C37H63O11, calcd. 683.4370). This was further
supported by pseudomolecular ion peaks in FAB‑MS at m/z 683
+ve) and 681 (−ve). The IR spectrum of 3 indicated the presence
(
Acknowledgements
−
1
of cyclopropyl methylene protons (2928 cm ), hydroxyl
!
−
1
−1
(
3408 cm ), and ester carbonyl (1728 cm ) functionalities. The
The authors are grateful to the Higher Education Commission, Is-
lamabad, Pakistan, for financial support to Saleem Jan through
the Indigenous Scholarship Scheme.
1
H NMR spectrum showed protons of cyclopropane methylene
(
δΗ 0.56, 0.26, J = 4.1 Hz, H -19), six tertiary methyls (δΗ 1.20,
2
1
.66, 1.71, 1.98, 1.34, 0.95 for CH -18, CH -26, CH -27, CH -28,
3 3 3 3
CH -29, CH -30, respectively), and one secondary methyl at δ
3
3
Η
1
.11 (d, J = 6.4 Hz, CH -21). Acetyl methyl protons appeared at δ
Conflict of Interest
3
Η
2.19 (s). Signal for an anomeric proton of the sugar moiety ap-
!
'
peared at δΗ 4.90 (d, J = 7.4 Hz, H-1 ), along with other signals of
sugar moiety. Large coupling of anomeric proton indicated a β-
linked sugar [12]. The C NMR spectrum of compound 3 showed
There is no conflict of interest issue.
1
3
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