6
H. Li et al.
that the aglycone of compound 2 was
gypsogenin [14,15], which was reported
from A. taipaiensis for the first time. The
copyranosyl gypsogenin 28-O-D-gluco-
pyranosyl-(1 ! 6)-b-D-glucopyranosyl
ester.
1
3
C NMR shifts of C-3 at d 80.4 and C-28
C
Four known saponins, namely 3-O-b-
D-xylopyranosyl-(1 ! 3)-a-L-rhamnopy-
ranosyl-(1 ! 2)-a-L-arabinopyranosyl
oleanolic acid (3) [16], 3-O-a-L-rhamno-
pyranosyl-(1 ! 2)-[b-D-glucopyranosyl-
(1 ! 4)]-a-L-arabinopyranosyl oleanolic
acid (4) [17], kizutasaponin K (5) [18],
at dC 177.6 implied that sugar linkages
were at both C-3 and C-28. The corre-
lations of H-3 with H-23 and H-5 observed
in the NOESY spectrum indicated the b-
configuration for the 3-O-sugar moiety
(
Figure 2). The presence of D-glucose, L-
rhamnose, and L-arabinose in a ratio of
:2:1 in compound 2 was established by
1
2
and hederacolchiside E (6) [19], respect-
ively, were identified by comparison of
their physical and spectroscopic data
(Supporting information) with those
reported in the literature. Saponin 5 was
obtained from A. taipaiensis for the first
time and the other three saponins were
firstly isolated from the aerial parts.
3
GC analysis of the corresponding tri-
1
methylsilylated derivatives. The H NMR
spectrum showed six anomeric proton
signals at d 5.87 (s, Rha I H-1), 6.13 (s,
H
Rha II H-1), 5.14 (d, J ¼ 6.4 Hz, Ara H-1),
4
.64 (d, J ¼ 6.2 Hz, Glc I H-1), 6.25 (d,
J ¼ 8.1 Hz, Glc II H-1), and 5.02 (d,
J ¼ 7.5 Hz, Glc III H-1) as well as the
corresponding anomeric carbons at dC
The cytotoxic activity of saponins 1–6
against human glioblastoma U251MG cells
was evaluated by MTT colorimetric assay
[20,21]. Nimustine hydrochloride was used
as a positive control. The IC50 value of each
compound was measured on the basis of
cell viability after 72 h treatment. As shown
in Table 3, two monodesmosidic saponins 3
and 4, which all possess a oligosaccharide
chain at C-3 and a free carboxylic acid at C-
28 of the aglycone, exhibited significant
cytotoxic activity with IC50 values of 1.56
and 9.14 mM, respectively, and saponin 3
was more potent, whereas the bisdesmosi-
dic saponins 1, 5, and 6 were all inactive
(IC50 . 100 mM). This suggested that the
presence of a free carboxylic group at C-28
is important for the cytotoxic activity.
Earlier studies on the cytotoxicity of
similar compounds have obtained the
same results [8,22]. However, bisdesmosi-
dic saponin 2 also showed marginal
cytotoxicity with an IC50 value of
80.62 mM. This may be ascribed to the
presence of the 23-CHO functionality.
1
02.9, 101.8, 106.6, 102.3, 95.8, and 105.0
1
3
observed in the C NMR spectrum. The b
and a anomeric configurations for the
glucose and rhamnose units respectively
were deduced by the same manner as for
compound 1. The arabinose unit was
determined to have an a anomeric
3
configuration on the basis of the J
H-1/H-2
4
6.4 Hz) value observed in the C form.
(
1
The assignments of NMR signals of the
oligosaccharide parts were achieved by a
combination of 2D NMR experiments. The
inter-glycosidic linkages of the sugar
chains and the linkages of the sugars
with the aglycone were established from
the key HMBC cross-peaks between H-1
of Glc I and C-3 of the aglycone, H-1 of
Rha I and C-2 of Glc I (dC 78.4), H-1
of Rha II and C-4 of Glc I (d 72.6), H-1 of
C
Ara and C-3 of Rha I (d 79.7), H-1 of Glc
C
III and C-6 of Glc II (d 69.3), and H-1 of
C
Glc II and C-28 of the aglycone, and the
conclusion was confirmed by the NOESY
correlations as shown in Figure 2. There-
fore, the structure of compound 2 was
elucidated as 3-O-a-L-arabinopyranosyl-
3.
Experimental
3.1 General experimental procedures
(
[
1 ! 3)-a-L-rhamnopyranosyl-(1 ! 2)-
UV spectra were measured on a Beckman
DU800 spectrometer (Beckman Coulter,
a-L-rhamnopyranosyl-(1 ! 4)]-b-D-glu-