9
06
J.-C. Yuan et al.
determined by correlations between b-
oriented protons H-20 at d 2.62 and H-23
at d 4.37 in ROESY spectrum (Figure 3).
By combined analysis of COSY, HSQC,
and HMBC spectra, all the proton and
carbon signals of 2 were fully assigned
and galactose moieties have b-configur-
ation [16]. Finally, by combined analysis
of COSY, HSQC, and HMBC spectra, all
the proton and carbon signals were fully
assigned (Tables 1 and 2) and the structure
of 3 was elucidated as (5b,17)-20-one-
pregn-16-ene-2b,3b-diol 3-O-b-D-gluco-
pyranosyl-(1 ! 2)-b-D-galactopyrano-
side, named timopregnane B.
(
Tables 1 and 2). Thus, the structure of 2
was elucidated as 5b-pseudo-spirost-
b,15a,23a-triol 3-O-b-D-glucopyrano-
3
syl-(1 ! 2)-b-D-galactopyranoside,
The structures of three known com-
pounds 4–6 isolated from this plant for the
first time were determined as 25R-
timosaponin BII [21], protodesgalactoti-
gonin [22], and timosaponin BII-a [23],
respectively, by comparison of their NMR
spectral data with those reported in the
literature.
named timosaponin Y.
Compound 3 was isolated as a white
amorphous powder. Its molecular formula
was determined to be C H O by HR-
3
3 52 13
2
ESI-MS at m/z 655.3533 [M 2 H] . ESI-
MS (negative mode) showed the ion peaks
2
at m/z 655 [M 2 H] , 493 [M 2 H–
2
62] , and 331 [M 2 H–162–162] ,
2
1
which attributed to loss of two hexoses.
Acid hydrolysis of 3 followed by GC
analysis of its derivative enabled the
identification of the two sugar units to be
3
.
Experimental
3.1 General experimental procedures
Optical rotations were measured with a
JASCO J-810 polarimeter (JASCO Corpor-
ation, Tokyo, Japan). The measurements of
IR were performed using Bruker Vertex 70
(Bruker Corporation, Karlsruhe, Germany).
1D and 2D NMR spectra in pyridine-d5
1
D-galactose and D-glucose. The H NMR
spectrum of 3 showed three singlet methyl
proton signals at d 0.96 (3H, s), 0.88 (3H,
s), and 2.21 (3H, s), one olefinic proton
signal at d 6.60 (1H, s), in addition to two
anomeric protons at d 4.98 (1H, d,
J ¼ 7.8 Hz) and 5.27 (1H, d, J ¼ 7.8 Hz).
UNITY-
solution were recorded on Varian
1
INOVA (600 MHz for H NMR and
13
150 MHz for C NMR) (Palo Alto, CA,
1
3
In the C NMR spectrum, a carbon signal
at d 196.3 (C-20) and two olefinic carbon
signals at d 144.6 and 155.5 were
observed, suggesting the existence of a
ketone group and one olefinic bond in the
molecule. The NMR data of 3 suggested
that it had the almost identical structure
with timopregnane A [20], except for the
ring A proportion. Compound 3 had one
more oxygen atom than timoprengane A
by comparing their molecular formula, and
the chemical shifts of C-1 (d 40.4), C-2 (d
USA), and the chemical shifts are expressed
in d (ppm) scale with TMS as an internal
reference. The HR-ESI-MS and ESI-MS
were recorded on Synapt Q/TOF MS
(Waters Corporation, Milford, MA, USA).
HPLC was undertaken with an Agilent 1100
(Agilent Technologies, Santa Clara, CA,
USA) equipped with Alltech 2000 ES
evaporative light-scattering detector (tem-
perature: 1108C, gas: 2.4l/min; Alltech
Corporation, Chicago, IL, USA) and Venu-
sil XBP C18 analytical column (4.6mm
£ 250 mm, 5 mm; Bonna-Agela, Tianjin,
China). Semi-preparativeHPLCseparations
were performed using the NP7000 module
(Hanbon Co. Ltd, Huaian, China) equipped
with a Shodex RID 102 detector (Showa
Denko Group, Tokyo, Japan) and a Vensiul
C18 column (8.0 mm £ 250 mm, ODS,
6
7.2), C-3 (d 81.9), C-9 (d 41.9), and C-10
d 37.2) inferred that 3 had a hydroxyl
(
group at C-2. The structure of ring A
proportion of 3 was further certified by
comparing its NMR data with those of
1
timosaponin N [14]. H NMR coupling
3
constants ( J . 7 Hz) for anomeric
protons revealed that both the glucose
1
,2