Nine new steroidal glycosides from the roots of Cynanchum stauntonii

Article abstract of DOI:10.1016/j.steroids.2012.10.007

Nine new steroidal glycosides, named as stauntosides C-K (2, 5, 7-10, 13, 14, and 16), along with seven known compounds (1, 3, 4, 6, 11, 12, and 15) were isolated from the 95% ethanol extract of the roots of Cynanchum stauntonii. The structures of these new compounds were elucidated on the basis of extensive spectroscopic analyses, mainly 1D and 2D NMR, and HRESI-MS, and qualitative chemical methods. Their significance in terms of the chemotaxonomy of C. stauntonii is discussed.

Full text of DOI:10.1016/j.steroids.2012.10.007

Steroids 78 (2013) 79–90
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Nine new steroidal glycosides from the roots of Cynanchum stauntonii
⇑
Jin-Qian Yu, An-Jun Deng, Hai-Lin Qin
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences
and Peking Union Medical College, Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 May 2012
Received in revised form 7 October 2012
Accepted 10 October 2012
Available online 2 November 2012
Nine new steroidal glycosides, named as stauntosides C–K (2, 5, 7–10, 13, 14, and 16), along with seven
known compounds (1, 3, 4, 6, 11, 12, and 15) were isolated from the 95% ethanol extract of the roots of
Cynanchum stauntonii. The structures of these new compounds were elucidated on the basis of extensive
spectroscopic analyses, mainly 1D and 2D NMR, and HRESI-MS, and qualitative chemical methods. Their
significance in terms of the chemotaxonomy of C. stauntonii is discussed.
Ó 2012 Elsevier Inc. All rights reserved.
Keywords:
Cynanchum stauntonii
Asclepiadaceae
Structure determination
Steroidal glycosides
Stauntosides C, D, E, F, G, H, I, J, and K
1. Introduction
type skeleton or the 14,15-secopregnane-type skeleton and were
given the trivial names stauntosides C–K, respectively, according
Cynanchum stauntonii (Decne.) Schltr. ex Levl., a perennial
medicinal herb from the family of Asclepiadaceae, is widely
distributed in south-central region of China. Along with another
species of the same genus, Cynanchum glaucescens (Decne.)
Hand.-Mazz., it has been used as antitussives and expectorants in
traditional Chinese medicine (TCM). Both of them are given the
Chinese name of ‘Bai Qian’ [1]. The main native organic compounds
isolated from Cynanchum species are steroids, especially the
steroidal saponins with aglycones assignable to either the normal
four-ring C21 steroid skeleton or the aberrant 13,14:14,15-diseco-
pregnane-type skeleton or the equally abnormal 14,15-secopregn-
ane-type skeleton, respectively [2,3]. These steroids have been
accepted, generally and chemotaxonomically, as the most impor-
tant and characteristic chemical constituents in Cynanchum
species. However, phytochemical investigation on the title plant
is very rare up to now and, to the best of our knowledge, with only
three papers having reported several steroids, including four by
our group 8 years ago [4]. The ongoing research aims at confirming
the phytotaxonomical basis from this plant through isolating and
elucidating more compounds, especially new ones. In this paper,
we describe nine new steroidal glycosides, compounds 2, 5, 7–10,
13, 14, and 16, and seven known compounds (1, 3, 4, 6, 11, 12 and
15) (Fig. 1), from the roots of C. stauntonii, and the evaluation of
their cytotoxic activities. The new steroidal glycosides contained
steroid aglycones with either the 13,14:14,15-disecopregnane-
to the priority order of isolation and their structural characteristics.
Also, this is the first isolation and characterization of the known
compounds 1, 4, 11, 12, and 15 from C. stauntonii. From a
phytochemical point of view, these new steroidal glycosides
isolated as major compounds from the title plant provided
additional evidence for the botanical classification of this genus
in Asclepiadaceae.
2. Experimental
2.1. General
Optical rotations were measured on a Perkin–Elmer 241 digital
polarimeter at 20 °C. IR spectra were recorded on a Nicolet 5700
spectrometer. 1D and 2D NMR spectra were taken on either a
Varian INOVA-500 spectrometer or a Varian NMR System-600
NMR spectrometer with tetramethylsilane as internal standard.
ESI-MS and HRESI-MS were obtained using an Agilent 1100 series
LC/MSD Trap SL mass spectrometer. Preparative HPLC was per-
formed on a Shimadzu LC-6AD system equipped with a SPD-10A
detector, and a reversed-phase C18 column (YMC-Pack ODS-A U
20 ꢀ 250 mm, 10
lm) was employed. GC analyses were conducted
on an Agilent 7890A instrument. Column chromatography (CC)
was undertaken over silica gel (200–300 mesh). TLC was carried
out with glass plate precoated silica gel G. Spots were visualized un-
der UV light and by spraying with 10% H2SO4 in 95% EtOH, followed
by heating. Acetonitrile used in preparative HPLC procedure was in
HPLC grade, and other solvents were of analytical grade.
⇑
Corresponding author. Tel.: +86 010 83172503; fax: +86 010 63017757.
E-mail addresses: fish@imm.ac.cn (J.-Q. Yu), denganjun@imm.ac.cn (A.-J. Deng),
qinhailin@imm.ac.cn (H.-L. Qin).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.10.007

80
J.-Q. Yu et al. / Steroids 78 (2013) 79–90
Fig. 1. The structures of compounds 1-16.
2.2. Plant material
Institute of Materia Medica, Chinese Academy of Medical Sciences
and Peking Union Medical College) according to the morphological
features. A voucher specimen (ID-S-2426) was deposited in the
Herbarium of Institute of Materia Medica, Chinese Academy of
Medical Sciences, Beijing, PR China.
The roots of C. stauntonii were collected from Tongbai County,
Henan Province of central China, in August, 2011. It was identified
by Associate Prof. Lin Ma (a savant in plant systematics from

Table 1
The 1H NMR chemical shifts of the sugar moieties of compounds 2, 5, 7–10, 13, 14, and 16 (d in ppm, J values in Hz).
H
2ab
b-
5cd
b-
7cb
b-
8cd
b-
9ed
b-
10ed
b- -the
13fd
b- -the
14cd
b- -the
16ed
b- -ole
D
-ole
D
-the
D
-the
D
-cym
D
-the
D
D
D
D
10
20
4.72 dd(2.1, 9.9)
1.27 m
4.27 d(7.0)
3.02
4.28 d(7.2)
3.03
4.62 br d(9.5)
1.25, 2.12
4.81 d(7.5)
3.93
4.78 d(7.5)
3.93
4.32 d(7.5)
3.39
4.32 d(7.5)
3.06
4.84 br d(8.5)
2.45, 1.80
2.24 m
3.18 m
2.96 m
3.25 m
1.23 d(6.0)
3.35 s
30
3.01
3.02
3.30
3.69
3.69
3.40
3.03
3.56
40
3.18
3.09
3.06
3.69
3.69
3.29
3.12
3.53
50
60
3.60
3.28
3.69
1.13 d(6.0)
3.31 s
3.69
1.46 d(5.0)
3.93 s
3.68
1.47 d(5.0)
3.92 s
3.56
3.29
1.14 d(6.0)
3.44 s
3.54
1.11*
3.43 s
1.12*
3.43 s
1.28*
3.62 s
1.45*
3.52 s
30-OCH3
b-
D-cym
b-
D-cym
b-
D-digt
b-
D-cym
b-
D-cym
b-D
-cym
b-
D-digt
b-D-digt
100
4.66 br d(9.5)
1.45, 2.05
3.40
4.75 br d(9.6)
2.01, 1.47
3.70
4.88 br d(9.5)
1.54, 1.89
3.98
3.07
3.27
5.31 br d(9.5)
2.36, 1.86
4.08
3.48
4.20
5.31 br d(9.0)
1.87, 2.35
4.08
4.90 br d(9.3)
2.13, 1.55
3.81
3.22
3.88
4.85 br d(8.0)
1.85, 1.53
4.03
3.13
3.71
5.43 br d(9.5)
2.40, 1.93
4.47
3.42
4.12 m
200
300
400
3.09
3.18
3.49 dd(2.5, 9.5)
4.22
500
3.30
3.69
600
1.11*
1.12*
1.17 d(6.0)
1.37 d(6.0)
3.61 s
1.37*
3.61 s
1.24 d(6.0)
1.12 d(6.5)
1.37 d(6.5)
300-OCH3
3.32 s
3.33 s
3.43*
b-
D-cym
b-
D-cym
a
-
L-cym
b-
D-cym
b-
D-cym
b-L
-dign
b-
D-cym
a-L-cym
1000
4.75 br d(9.0)
1.46, 2.01
3.70
4.69 br d(10.2)
2.13, 1.47
3.61
4.81 br s
1.69 td (14.5, 3.5), 2.07
3.46
3.19
3.98
1.09 d(6.5)
3.29 s
5.08 br d(9.5)
2.41, 1.74
3.87
3.45
4.21
5.08 br d(9.0)
1.74, 2.43
3.94
4.68 br d(9.9)
2.25, 1.63
3.62
4.70 dd(10.0, 2.0)
2.07, 1.51
3.51
3.06
3.62
5.07 br s
2.36, 1.83
3.70 m
3.60
2000
3000
4000
3.08
3.17
3.45 dd(2.5, 10.0)
4.21
3.20
5000
3.68
3.72
3.35
4.47
6000
1.11*
1.12*
1.37 d(6.0)
3.53 s
1.37*
3.53 s
1.28*
1.11 d(6.5)
3.35 s
1.43*
3000-OCH3
3.35 s
3.32 s
3.43*
3.38 s
a
-
L
-dign
a
-
L
-dign
a-L-cym
5.22 br d(3.5)
2.08, 2.39
10000
20000
4.90 d(3.6)
1.65 dd (12.0, 4.2)
1.78 td (12.6, 3.6)
3.41
5.22 d(2.5)
2.39, 2.08
30000
3.85
4.07
4.31
1.57 d(7.0)
3.31 s
3.86
3.69
40000
3.63
50000
3.85
4.32 dd(6.5, 13.0)
1.56 d(7.5)
3.32 s
60000
1.12*
30000-OCH3
3.23 s
a: in CD3COCD3; b: 600 MHz; c: in DMSO-d6; d: 500 MHz; e: in C5D5N; f: in CDCl3.
cym, cymaropyranosyl; dign, diginopyranosyl; digt, digitoxopyranosyl; ole, oleandropyranosyl; the, thevetopyranosy.
*
Overlapped.

82
J.-Q. Yu et al. / Steroids 78 (2013) 79–90
2.3. Extraction and isolation
(F C1–C7, also according to the detection of TLC). Fraction C2 was
subjected to CC over silica gel with petroleum ether/EtOAc (4:1)
as eluants resulting in the isolation of compound 11 (5 mg). Frac-
tion C3 was subjected to CC over silica gel with petroleum ether/
acetone (5:1) and petroleum ether/acetone (3:1) as eluants,
respectively, resulting in the isolation of compounds 12 (10 mg)
and 13 (16 mg). Fraction C4 was applied to preparative HPLC
system [mobile phase: CH3OH/H2O (70:30, v/v); flow rate:
5 mL minꢁ1; UV detection at 280 nm] resulting in the isolation of
compound 14 (38 mg). Fraction C5 was applied to Flash C18 column
chromatography eluted with CH3OH/H2O (40% ? 100%) to give six
subfractions (FC5-1–FC5-6). Fraction C5-2 was applied to prepara-
tive HPLC system [mobile phase: CH3CN/H2O (40:60, v/v); flow
The dried-up and pulverized roots (30 kg) of C. stauntonii were
extracted three times under reflux with 95% EtOH. The combined
ethanolic solution was concentrated in vacuo to yield a dark-
brown residue (ca. 5000 g). The residue was suspended in 80%
aqueous ethanol (ca. 10000 mL) and then extracted with, firstly,
petroleum ether, and secondly, EtOAc, respectively and succes-
sively, each for several times until the upper layer was transparent,
in separatory funnel. The combined EtOAc solution was washed
with 5% aqueous solution of NaHCO3 (3ꢀ 1000 mL) and then H2O
(2ꢀ 1000 mL), respectively, to pH 7. After removal of the organic
solvent, 190 g of brown residue was obtained. This resulting resi-
due was fractionated by CC over silica gel eluted with gradient sol-
vents of CHCl3–CH3OH (100:0 ? 1:1) to yield 13 fractions
(designated as fraction A to M) according to their TLC profiles. Frac-
tion B (27 g) was further separated by CC over silica gel using gra-
dient solvents of petroleum ether/EtOAc (25:1 ? 1:1) as eluents to
yield eight further subfractions (F B1–B8, also according to the
detection of TLC). Fraction B2 was subjected to CC over silica gel
with petroleum ether/EtOAc (7:3) and petroleum ether/EtOAc
(4:1) as eluants, respectively, resulting in the isolations of com-
pounds 1 (5 mg) and 2 (40 mg). Fraction B3 was applied to pre-
parative HPLC system [mobile phase: CH3OH/H2O (75:25, v/v);
flow rate: 5 mL minꢁ1; UV detection at 210 nm] resulting in the
isolation of compounds 3 (220 mg), 4 (10 mg), and 5 (30 mg). Frac-
tion B4 was subjected to preparative HPLC system [mobile phase:
CH3OH/H2O (70:30, v/v); flow rate: 5 mL minꢁ1; UV detection at
210 nm] resulting in the isolation of compounds 6 (13 mg) and 7
(24 mg). Fraction B5 was applied to preparative HPLC system [mo-
bile phase: CH3CN/H2O (45:55, v/v); flow rate: 5 mL minꢁ1; UV
detection at 210 nm] resulting in the isolation of compound 8
(40 mg). Fraction B6 was subjected to preparative HPLC system
rate: 5 mL minꢁ1
; UV detection at 210 nm] resulting in the
isolation of compound 15 (25 mg). Fraction C6 was applied to Flash
C18 column chromatography eluted with CH3OH/H2O (40% ?
100%) to give six subfractions (FC6-1–FC6-6). Fraction C6-3 was
applied to preparative HPLC system [mobile phase: CH3CN/H2O
(45:55, v/v); flow rate: 5 mL minꢁ1; UV detection at 280 nm]
resulting in the isolation of compound 16 (40 mg).
The seven known compounds, hirundoside
glaucogenin 3-O- -cymaropyranosyl-(1 ? 4)-b-
ransyl-(1 ? 4)-b- -canaropyranoside (3) [6], cynatratoside B (4)
A
(1) [5],
C
a-
L
D-digtoxopy-
D
[7], stauntoside B (6) [8], 2,4-dihydroxyacetophenone (11) [9],
14-hydroxyandrosta-4,6,15-triene-3,17-dione (12) [10], and
glaucogenin C mono-D-thevetoside (15) [11] were identified by
comparison of their spectroscopic data (1H and 13C NMR, MS) with
the literature values.
2.3.1. Stauntoside C (2)
White lamellae (CH3OH–CHCl3), ½a D20
ꢂ
+276.3 (c = 1.04, CHCl3,
20 °C). IR (KBr) mmax: 3498, 2936, 1681, 1325, 1185, 1101, 1063,
980, 861 cmꢁ1. ESI-MS (positive mode) m/z: 509.2 [M+Na]+. HRES-
I-MS (positive mode) m/z: 509.2520 (Diff 2.21 ꢀ 10ꢁ6) [M+Na]+,
calcd for C28H38O7Na, 509.2510. 1H NMR (600 MHz, CD3COCD3)
data for aglycone: d 0.83 (3H, s, H-19), 1.45 (3H, s, H-21), 2.06
(ov, H-9), 2.87 (1H, d, J = 7.2 Hz, H-17), 3.77 (1H, d, J = 8.4 Hz, H-
18a), 3.84 (1H, dd, J = 10.9, 4.5 Hz, Hb-15), 4.08 (1H, d, J = 8.4 Hz,
[mobile phase: CH3CN/H2O (38:62, v/v); flow rate: 5 mL minꢁ1
;
UV detection at 254 nm] resulting in the isolation of compound 9
(20 mg). Fraction B7 was applied to preparative HPLC system [mo-
bile phase: CH3OH/H2O (70:30, v/v); flow rate: 5 mL minꢁ1; UV
detection at 254 nm] resulting in the isolation of compound 10
(20 mg). Fraction C (68 g) was further separated by CC over silica
gel using gradient solvents of petroleum ether/EtOAc
(25:1 ? 1:1) as eluents to yield seven further subfractions
H-18b), 4.08 (1H, br d, J = 10.9 Hz, H -15), 4.31 (1H, t, J = 8.4 Hz,
a
H-3), 4.84 (1H, m, H-16), 5.48 (1H, br s, H-4), 5.80 (1H, d,
J = 9.6 Hz, H-6), 6.35 (1H, d, J = 9.6 Hz, H-7). 1H NMR (600 MHz,
Table 2
The 13C NMR chemical shifts of the aglycone moieties of compounds 2, 5, 7-10, 13, 14, and 16.
cb
C
2ab
5cd
5be
7
7be
8cd
8be
9ed
10ed
13fd
14cd
14be
16ed
1
2
3
4
5
6
7
8
34.1
28.0
35.9
29.2
77.6
38.1
140.0
120.1
27.6
39.8
52.5
38.1
23.4
29.4
117.8
174.9
66.9
74.8
55.4
143.4
17.7
113.6
24.5
37.0
30.1
78.8
39.0
140.6
120.4
30.0
40.7
53.2
38.7
23.9
28.4
118.5
175.5
67.8
75.5
56.2
143.7
17.9
114.4
24.8
36.5
29.3
76.8
38.0
139.4
119.3
29.0
37.3
30.1
36.9
29.5
76.2
38.1
139.4
119.5
29.1
39.9
51.3
37.2
26.3
146.5
129.4
178.7
70.7
37.3
30.2
76.9
39.0
140.2
119.8
30.0
41.0
52.2
38.0
27.2
146.9
129.9
179.3
71.3
77.3
54.5
167.5
20.0
33.7
28.9
77.6
38.9
164.9
126.5
187.0
131.0
162.3
42.5
22.4
27.3
58.2
105.7
72.2
79.4
59.2
74.9
23.0
36.6
27.2
75.5
124.3
143.8
130.2
131.4
75.8
55.8
37.4
19.3
34.5
58.5
111.3
73.6
83.2
69.5
76.2
21.7
118.1
22.9
36.2
29.7
78.1
38.5
140.3
120.3
25.3
100.7
44.6
37.6
19.9
31.7
53.3
151.8
72.4
83.7
63.4
76.4
18.6
117.7
22.5
32.7
27.0
74.6
124.5
143. 7
125.0
122.0
107.0
43.4
34.8
19.7
30.0
54.0
154.9
71.2
85.6
61.0
76.5
33.5
27.7
75.4
125.1
144.5
125.7
122.6
108.2
44.2
35.6
20.4
33.6
28.0
74.9
125.1
145.3
125.9
122.9
108.4
44.8
36.1
20.8
31.3
55.3
155.9
72.3
86.7
62.5
77.6
18.0
77.5
38.9
140.1
119.7
30.0
40.9
52.2
38.0
27.2
146.9
129.9
179.3
71.3
78.1
54.5
167.5
20.0
74.6
124.8
144.9
125.7
122.8
108.1
44.2
35.6
20.5
40.1
9
51.2
37.1
26.2
146.4
129.3
178.6
70.6
77.1
53.3
166.7
19.6
113.1
23.1
10
11
12
13
14
15
16
17
18
19
20
21
30.8
30.8
54.8
155.3
72.1
86.2
62.0
77.4
17.7
118.4
22.6
54.8
155.4
72.1
86.2
62.0
77.4
17.7
118.4
22.6
77.2
53.4
166.9
19.6
113.2
23.2
17.4
117.7
22.4
118.7
22.7
113.4
23.5
113.4
23.5
118.5
23.1
a: in CD3COCD3; b: 150 MHz; c: in DMSO-d6; d: 125 MHz; e: in C5D5N; f: in CDCl3.

J.-Q. Yu et al. / Steroids 78 (2013) 79–90
83
Table 3
The 13C NMR chemical shifts of the sugar moieties of compounds 2, 5, 7-10, 13, 14 and 16.
cb
C
2ab
b-
5cd
b-
5be
b-
7
7be
b-
8cd
b-
8be
b-
9ed
b-
10ed
b- -the
13fd
b- -the
14cd
b- -the
14be
b- -the
16ed
b- -ole
D
-ole
D
-the
D
-the
b-
D-the
D
-the
D
-cym
D
-cym
D
-the
D
D
D
D
D
10
99.3
37.3
81.7
76.7
72.7
18.4
56.9
100.8
73.0
84.3
82.0
69.6
18.1
59.3
102.2
74.7
85.8
83.2
70.9
18.7
60.5
100.7
72.9
84.2
81.6
70.0
17.9
59.2
102.1
74.7
85.8
82.7
71.6
18.6
60.5
96.9
36.6
78.0
82.2
68.1
18.1
56.5
98.0
37.9
79.2
83.1
69.2
18.6
57.4
102.4
74.5
85.8
82.6
71.7
18.4
60.6
103.2
74. 6
85.8
82.7
71.7
18.7
60.5
100.7
73.7
84.1
81.6
70.7
18.3
59.8
101.8
72.9
84.4
81.9
70.2
18.4*
59.3
103.3
74.6
85.8
83.2
71.7
18.0
60.5
98.8
37.9
79.2
83.1
71.7
18.8
57.4
20
30
40
50
60
30-OCH3
b-
D
-cym
b-
D
-cym
b-
D-cym
b-
D-cym
b-
D-digt
b-
D-digt
b-
D-cym
b-
D
-cym
b-
D-cym
b-
D-digt
b-
D-digt
b-D-digt
100
99.6
35.6
77.4
81.8
70.1
18.9
51.8
100.5
36.5
78.2
82.7
71.6
19.0
58.7
97.8
35.5
76.8
81.9
68.2
18.1*
57.7
98.8
37.1
77.5
83.2
69.3
18.7
58.9
97.5
38.1
66.6
80.1
70.5
18.3
98.4
38.5
67.9
80.8
71.7
18.8
98.8
37.1
78.7
83.2
69.3
18.6
58.9
98.8
37.1
78.1
83.3
69.3
18.6
58.9
98.6
35.4
77.2
82.4
68.8
18.0
57.9
97.9
38.3
66.4
81.6
67.8
18.0*
98.9
39.1
67.6
82.9
68.8
18.7
98.5
38.5
67.9
80.8
69.2
18.6
200
300
400
500
600
300-OCH3
b-
D
-cym
b-
D
-cym
b-
D-cym
b-
D-cym
a
-
L-cym
a
-
L-cym
b-
D-cym
b-
D
-cym
b-
L
-dign
b-
D-cym
b-
D-cym
a-L-cym
1000
97.9
35.0
76.9
72.9
68.3
18.0
57.8
98.8
35.8
78.1
74.1
69.3
18.4
58.1
99.2
34.1
76.3
80.9
68.2
17.9*
56.7
100.2
35.1
77.3
82.2
69.3
18.6
57.3
97.6
31.3
75.4
71.5
66.0
18.0
56.2
98.6
32.2
76.6
72.7
67.2
18.4
56.8
100.2
35.1
77.5
82.2
69.3
18.6
57.3
100.2
35.1
77.5
82.2
69.3
18.4
57.3
99.5
33.7
77.4
72.4
71.3
18.1
57.2
99.0
34.7
77.6
72.8
69.7
17.9*
57.8
99.8
35.6
78.8
74.1
70.9
18.5
58.0
98.4
32.2
76.6
72.7
67.1
18.4
56.8
2000
3000
4000
5000
6000
3000-OCH3
a-
L-dign
a-
L
-dign
a
-L
-dign
a-L-cym
10000
99.7
29.8
74.5
66.5
66.0
17.1
54.4
101.1
31.0
74.7
67.6
67.7
18.4
55.0
101.1
30.9
75.8
67.6
67.7
17.7
55.0
101.1
30.9
75.8
71.7
67.7
17.7
55.0
20000
30000
40000
50000
60000
30000-OCH3
a: in CD3COCD3; b: 150 MHz; c: in DMSO-d6; d: 125 MHz; e: in C5D5N; f: in CDCl3.
cym, cymaropyranosyl; dign, diginopyranosyl; digt, digitoxopyranosyl; ole, oleandropyranosyl; the, thevetopyranosyl.
*
Overlapped.
CD3COCD3) data of the sugar moiety: see Table 1. 13C NMR
(150 MHz, CD3COCD3): see Tables 2 and 3.
13C NMR (150 MHz, DMSO-d6 and 150 MHz, C5D5N): see Tables 2
and 3.
2.3.4. Stauntoside F (8)
2.3.2. Stauntoside D (5)
White amorphous powder (CH3OH–CHCl3), ½a D20
ꢁ91.8 (c = 1.01,
ꢂ
White amorphous powder (CH3OH–CHCl3), ½a D20
ꢂ
+20.1 (c = 1.01,
CH3OH, 20 °C). IR (KBr) mmax: 3494, 2934, 1756, 1726, 1660, 1450,
CH3OH, 20 °C). IR (KBr) mmax: 3483, 2932, 1735, 1652, 1452, 1380,
1373, 1162, 1059, 909, 815 cmꢁ1. ESI-MS (positive mode) m/z:
815.4 [M+Na]+. HRESI-MS (positive mode) m/z: 815.3858 (Diff
3.75 ꢀ 10ꢁ6) [M+Na]+, calcd for C41H60O15Na, 815.3878. 1H NMR
(500 MHz, DMSO-d6) data for aglycone: d 1.00 (3H, s, H-19), 1.57
(3H, s, H-21), 3.46 (1H, m, H-3), 3.86 (1H, dd, J = 10.5, 4.5 Hz, Hb-
1308, 1161, 1062, 1004, 868, 606 cmꢁ1. ESI-MS (positive mode) m/
z 831.4 [M+Na]+. HRESI-MS (positive mode) m/z: 831.4137 (Diff
0.19 ꢀ 10ꢁ6) [M+Na]+, calcd for C42H64O15Na, 831.4137. 1H NMR
(500 MHz, DMSO-d6) data for aglycone: d 0.86 (3H, s, H-19), 1.43
(3H, s, H-21), 3.35 (1H, ov, H-17), 3.49 (1H, m, H-3), 3.62 (1H, m,
15), 4.48 (1H, dd, J = 10.5, 7.5 Hz, H -15), 5.33 (1H, br s, H-6),
a
Hb-15), 4.16 (1H, t, J = 8.0 Hz, H -15), 5.26 (1H, dd, J = 8.0, 7.5 Hz,
a
5.62 (1H, m, H-16), 6.03 (1H, dd, J = 5.0, 13.0 Hz, H-12). 1H NMR
(500 MHz, DMSO-d6) data of the sugar moiety: see Table 1. 13C
NMR (125 MHz, DMSO-d6 and 150 MHz, C5D5N): see Tables 2
and 3.
H-16), 5.38 (1H, m, H-6), 6.40 (1H, s, H-18). 1H NMR (500 MHz,
DMSO-d6) data of the sugar moiety: see Table 1. 13C NMR
(125 MHz, DMSO-d6 and 150 MHz, C5D5N): see Tables 2 and 3.
2.3.3. Stauntoside E (7)
2.3.5. Stauntoside G (9)
White amorphous powder (CH3OH–CHCl3), ½a D20
ꢂ
ꢁ35.1 (c = 1.00,
White amorphous powder (CH3OH–CHCl3), ½a D20
ꢁ3.6 (c = 0.55,
ꢂ
CH3OH, 20 °C). IR (KBr)
m
max: 3414, 2934, 1754, 1725, 1658, 1449,
CH3OH, 20 °C). IR (KBr) mmax: 3454, 2933, 1659, 1615, 1451,
1369, 1165, 1062, 908, 817 cmꢁ1. ESI-MS (positive mode) m/z:
989.6 [M+Na]+. HRESI-MS (positive mode) m/z: 989.4727 (Diff
1.00 ꢀ 10ꢁ6) [M+Na]+, calcd for C49H74O19Na, 989.4717. 1H NMR
(600 MHz, DMSO-d6) data for aglycone: d 1.01 (3H, s, H-19), 1.57
(3H, s, H-21), 3.71 (1H, m, H-3), 3.87 (1H, m, Hb-15), 4.48 (1H,
1377, 1157, 1061, 939 cmꢁ1. ESI-MS (positive mode) m/z: 989.5
[M+Na]+. HRESI-MS (positive mode) m/z: 989.4701 (Diff
1.68 ꢀ 10ꢁ6) [M+Na]+, calcd for C49H74O19Na, 989.4717. 1H NMR
(500 MHz, C5D5N) data for aglycone: d 1.10 (3H, s, H-19), 1.60
(3H, s, H-21), 2.99 (1H, d, J = 7.0 Hz, H-17), 3.72 (1H, dd, J = 10.5,
3.5 Hz, Hb-15), 3.89 (1H, d, J = 9.5 Hz, H-18a), 3.94 (1H, m, H-3),
dd, J = 10.8, 7.2 Hz, H -15), 5.33 (1H, br s, H-6), 5.62 (1H, ddd,
a
J = 7.8, 7.2, 4.8 Hz, H-16), 6.04 (1H, dd, J = 4.8, 12.6 Hz, H-12). 1H
NMR (600 MHz, DMSO-d6) data of the sugar moiety: see Table 1.
4.26 (1H, m, H-16), 4.31 (1H, t, J = 10.5 Hz, H -15), 5.23 (1H, d,
a
J = 9.5 Hz, H-18b), 6.23 (1H, br s, H-6). 1H NMR (500 MHz, C5D5N)

84
J.-Q. Yu et al. / Steroids 78 (2013) 79–90
data of the sugar moiety: see Table 1. 13C NMR (125 MHz, C5D5N):
see Tables 2 and 3.
evaporated under vacuum, then re-diluted with water and re-
evaporated in vacuo repeatedly to eliminate the surplus HCl and
furnish a final neutral residue. The residue was analyzed by TLC
with silica gel G as adsorbents, 10% H3PO4ꢃ12MoO3 (phosphomo-
lybdic acid hydrate) in 95% EtOH as detection reagent for spraying,
followed by heating the plate to develop the colors, and solvent A,
CHCl3–CH3OH (8:1), and solvent B, EtOAC–acetone (2.5:2) as sol-
vent systems, respectively, for development of sugars. The Rf val-
ues of digitoxose, thevetose, oleandrose, and cymarose were
determined, by interactive comparison among the known com-
pounds 3, 4, and 15, in the order of 0.84, 0.70, 0.38, and 0.34 over
solvent A, and of 0.88, 0.75, 0.45, and 0.40 over solvent B,
respectively.
2.3.6. Stauntoside H (10)
White amorphous powder (CH3OH-CHCl3), ½a D20
ꢂ
+2.5 (c = 0.96,
CH3OH, 20 °C) IR (KBr) mmax: 3445, 2934, 1623, 1545, 1452, 1379,
1366, 1164, 1062, 891 cmꢁ1. ESI-MS (positive mode) m/z: 991.5
[M+Na]+. HRESI-MS (positive mode) m/z: 991.4845 (Diff
2.99 ꢀ 10ꢁ6) [M+Na]+, calcd for C49H76O19Na, 991.4873. 1H NMR
(500 MHz, C5D5N) data for aglycone: d 1.38 (3H, s, H-19), 1.61
(3H, s, H-21), 2.26 (1H, dd, J = 7.0, 12.0 Hz, H-9), 3.15 (1H, d,
J = 7.5 Hz, H-17), 3.77 (1H, dd, J = 10.5, 4.0 Hz, Hb-15), 4.06 (1H,
ov, H-18a), 4.25 (1H, t, J = 10.5 Hz, H -15), 4.46 (1H, t, J = 8.0 Hz,
a
H-3), 5.17 (1H, ov, H-18b), 5.17 (1H, m, H-16), 5.68 (1H, s, H-4),
6.12 (1H, d, J = 10.0 Hz, H-6), 6.44 (1H, d, J = 10.0 Hz, H-7). 1H
NMR (500 MHz, C5D5N) data of the sugar moiety: see Table 1. 13C
NMR (125 MHz, C5D5N): see Tables 2 and 3.
2.5. Determination of the absolute configurations of monosaccharides
The absolute configurations of
D-digitoxose, L-cymarose, D-
thevetose, and -oleandrose were determined as per the method
published by Hara et al. [12]. The monosaccharides obtained on
acid hydrolysis, as described above, were dissolved in pyridine
D
2.3.7. Stauntoside I (13)
White amorphous powder (CH3OH-CHCl3), ½a D20
ꢂ
+43.9 (c = 0.98,
CHCl3, 20 °C). IR (KBr) mmax: 3451, 2933, 1681, 1380, 1163, 1062,
and reacted with -cysteine methyl ester hydrochloride at 60 °C
L
1006, 868 cmꢁ1. ESI-MS (positive mode) m/z: 815.5 [M+Na]+.
for 1 h. Equal volume of acetic anhydride was added and heating
was carried out for another 1 h. Acetylated thiazolidine derivatives
were injected into GC system. The absolute configurations of the
sugars were determined by comparing the retention times with
those of acetylated thiazolidine derivatives synthesized from the
known sugars obtained through acid hydrolysis of the known com-
HRESI-MS (positive mode) m/z: 815.4188 (Diff 0.09 ꢀ 10ꢁ6
)
[M+Na]+, calcd for C42H64O14Na, 815.4188. 1H NMR (500 MHz,
CDCl3) data for aglycone: d 0.82 (3H, s, H-19), 1.52 (3H, s, H-21),
3.58 (1H, m, H-3), 3.85 (1H, dd, J = 10.8, 4.5 Hz, Hb-15), 3.90 (2H,
s, H-18), 4.18 (1H, br d, J = 10.8 Hz, H -15), 4.74 (1H, m, H-16),
a
5.33 (1H, br s, H-6). 1H NMR (500 MHz, CDCl3) data of the sugar
moiety: see Table 1. 13C NMR (125 MHz, CDCl3): see Tables 2 and 3.
pounds 3, 4, and 15 (Also, the retention times of
D-digitoxose, L-
cymarose, -thevetose, and -oleandrose were determined by
D
D
interactive comparison among the known compounds 3, 4, and
15. GC conditions in the test: column, HP-5, 30 m ꢀ 0.25 mm,
2.3.8. Stauntoside J (14)
White amorphous powder (CH3OH–CHCl3), ½a D20
ꢂ
+20.6 (c = 1.01,
0.25 lm; detection, FID; carrier gas, N2; injection temperature,
CH3OH, 20 °C). IR (KBr) mmax: 3485, 2934, 1677, 1378, 1161, 1065,
250 °C, detection temperature, 280 °C, column temperature,
150 °C (0 min), 10 °C/min to 250 °C (20 min). tR -digitoxose
13.09 min, tR -cymarose 13.46 min, tR -thevetose 16.07 min,
and tR -oleandrose 18.52 min).
867 cmꢁ1. ESI-MS (positive mode) m/z: 799.5 [M+Na]+. HRESI-MS
(positive mode) m/z: 799.3892 (Diff 2.02 ꢀ 10ꢁ6) [M+Na]+, calcd
for C41H60O14Na, 799.3875. 1H NMR (500 MHz, DMSO-d6) data
for aglycone: d 0.75 (3H, s, H-19), 1.42 (3H, s, H-21), 1.97 (1H,
ov, H-9), 2.87 (1H, d, J = 8.0 Hz, H-17), 3.64 (1H, d, J = 8.0 Hz, H-
18a), 3.80 (1H, dd, J = 11.0, 4.5 Hz, Hb-15), 3.89 (1H, d, J = 8.0 Hz,
D
L
D
D
Not all of the monosaccharides involved in this paper were de-
tected by GC method because of the lack of reference sugars, but,
from the results of the typical monosaccharides, it can be con-
cluded that the absolute configurations of the monosaccharides
composed of the sugar units can be really suggested by comparison
of their spectroscopic data with those reported in the literature.
H-18b), 4.04 (1H, br d, J = 11.0 Hz, H -15), 4.24 (1H, m, H-3), 4.84
a
(1H, m, H-16), 5.49 (1H, s, H-4), 5.80 (1H, d, J = 9.5 Hz, H-6), 6.30
(1H, d, J = 9.5 Hz, H-7). 1H NMR (500 MHz, DMSO-d6) data of the
sugar moiety: see Table 1. 13C NMR (125 MHz, DMSO-d6): see Ta-
bles 2 and 3.
2.6. Cytotoxicity assay
2.3.9. Stauntoside K (16)
Compounds 1–16 were tested for cytotoxicity against HCT-8
(human colon cancer cell line), Bel-7402 (human hepatoma cancer
cell line), BGC-823 (human gastric cancer cell line), A549 (human
lung epithelial cell line), and A2780 (human ovarian cancer cell
line) by means of the MTT method as described in the literature
[13].
White amorphous powder (CH3OH–CHCl3), ½a D20
ꢂ
+24.9 (c = 1.07,
CH3OH, 20 °C). IR (KBr) mmax: 3461, 2933, 1679, 1377, 1158, 1059,
989, 866 cmꢁ1. ESI-MS (positive mode) m/z:783.4 [M+Na]+. HRESI-
MS (positive mode) m/z: 783.3930 (Diff 0.67 ꢀ 10ꢁ6) [M+Na]+,
calcd for C41H60O13Na, 783.3926. 1H NMR (500 MHz, C5D5N) data
for aglycone: d 0.84 (3H, s, H-19), 1.57 (3H, s, H-21), 2.17 (1H,
ov, H-9), 2.78 (1H, d, J = 8.0 Hz, H-17), 3.82 (1H, dd, J = 11.0,
3. Results and discussion
4.0 Hz, Hb-15), 4.04 (2H, m, H-18), 4.28 (1H, br d, J = 11.0 Hz, H -
a
15), 4.53 (1H, m, H-3), 4.80 (1H, m, H-16), 5.75 (1H, s, H-4), 6.05
(1H, d, J = 9.5 Hz, H-6), 6.70 (1H, d, J = 9.5 Hz, H-7). 1H NMR
(500 MHz, C5D5N) data of the sugar moiety: see Table 1. 13C NMR
(125 MHz, C5D5N): see Tables 2 and 3.
All nine new compounds were obtained as white lamellae or
amorphous powder, and showed positive Libermann-Burchard
and Keller-Kiliani reactions, suggesting their glycosidic steroidal
category with 2-deoxysugar units existing in their sugar moieties
[14].
2.4. Acid hydrolysis of 2–5, 7–10, and 13–16
Each solution of 6 mg of compounds 2–5, 7–10, and 13–16 was
refluxed within 10% HCl (3 ml) at 75 °C for 2.5 h. After cooling, the
reaction mixture was extracted thoroughly with CHCl3, the CHCl3
layer was washed with water, and then the water fraction was
combined with the original aqueous layer. The aqueous layer was
3.1. Stauntoside C (2)
Compound 2 was determined to possess the molecular formula
C
28H38O7 by its pseudomolecular ion peak at m/z 509.2520
[M+Na]+ in the positive HRESI-MS experiment. The IR spectrum

J.-Q. Yu et al. / Steroids 78 (2013) 79–90
85
showed the absorption bands for hydroxy (3498 cmꢁ1) and olefinic
(1681 cmꢁ1) groups. The 1H, 13C, and DEPT NMR spectroscopic data
of 2 (see Section 2 and Tables 1–3) revealed the diagnostic signals
of steroidal glycoside, with an aglycone of 14,15-secopregnane-
type skeleton and one 2,6-dideoxypyranose unit being exhibited.
Comparison of 1H and 13C NMR spectroscopic data of 2 with those
of amplexicogenin A in amplexicoside G, a known aglycone of ste-
roidal glycoside isolated from the roots of Cynanchum amplexicaule
[14], as well as the information obtained in HSQC experiments,
demonstrated that most signals of 2 were superimposable to its
counterparts in amplexicogenin A, except for the absence in com-
pound 2 of the characteristic signal for the oxymethine at CH-2 in
amplexicogenin A, with the dehydroxylation shifts observed at C-1
(ꢁ7.8), C-2 (ꢁ41), and C-3 (ꢁ9.5) in the 13C NMR spectrum of 2.
Also, by comparing to amplexicogenin A, the HMBC experiment
(data not shown) confirmed the connectivity in 2 which was con-
firmed by the positive ESI-MS/MS spectrum exhibiting the frag-
ment ion peaks at m/z 347.1 [M-18-ole+Na]+. Compound 2 was
subjected to acid hydrolysis as described above in Sections 2.4
and 2.5 and the obtained monosaccharide was determined to be
Fig. 2. Observed NOE correlations in the oligosaccharide moiety of stauntoside D
(5).
by the splitting patterns and coupling constants of the above
mentioned anomeric proton signals, and by the NOE correlations
of H-10ax and H-30ax and H-50ax, of H-100 and H-200 and H-500
,
ax
eq
ax
of H-1000 and H-2000eq, among others (Fig. 2), in the NOESY spec-
ax
trum. The D-type absolute configurations of three sugars were all
speculated by comparing the NMR data of the sugars with those
of corresponding sugars of cynaforroside Q [15], amplexicoside E
[14], amplexicoside F [14], and
D-cymaropyranose group [16].
These conclusions about the absolute configurations of the mono-
saccharides were confirmed by acid hydrolysis as described above
in Sections 2.4 and 2.5, which not only gave one D-thevetopyranose
and another kind of sugar unit, but also confirmed that the abso-
lute configurations of the monosaccharides speculated by compar-
ison of their spectroscopic data with those reported are really
consistent with reality. Also, this determination is because of the
very common and limited kinds of the sugars. Because of the lack
oleandropyranose in the
D-form by GC analysis. Therefore, com-
pound 2 was elucidated as deoxyamplexicogenin A 3-O-b-
D
-olean-
dropyranoside (Fig. 1), and was given the trivial name of
stauntoside C.
3.2. Stauntoside D (5)
The positive HRESI-MS of 5 gave a pseudomolecular ion peak at
m/z 831.4137 [M+Na]+, corresponding to the molecular formula
of reference substance,
determined in the GC test. The sugar sequence of 5 was demon-
strated by HMBC correlations from dH 4.75 (H-1000 of b-
-cymaro-
pyranose) to dC 81.8 (C-400 of b-
-cymaropyranose), from dH 4.66
(H-100 of b- -cymaropyranose) to dC 82.0 (C-40 of b-
-thevetopyra-
nose), and from dH 4.27 (H-10 of b-
-thevetopyranose) to dC 77.6
D-cymaropyranose units could not be
C42H64O15. The IR spectrum showed the absorption bands for hy-
D
droxy (3483 cmꢁ1), carbonyl (1735 cmꢁ1), and olefinic
(1652 cmꢁ1) groups. The 1H NMR spectrum of 5 revealed the diag-
nostic signals of steroidal glycoside, with a 13,14:14,15-diseco-
pregnane-type skeleton aglycone being exhibited typically by
two tertiary methyls and one methyleneoxy group (see experimen-
tal section), and with three 6-deoxypyranoses being shown by
three anomeric proton signals at d 4.27 (1H, d, J = 7.0 Hz, H-10),
4.66 (1H, d, J = 9.5 Hz, H-100), and 4.75 (1H, d, J = 9.0 Hz, H-1000),
which correlated to the corresponding anomeric carbon signals at
dC 100.8 (C-10), 99.6 (C-100), and 97.9 (C-1000), respectively, in the
HSQC spectrum, and three secondary methyls at d 1.11 (9H, ov,
H-60, 600, and 6000) (Table 1). In addition, three methoxyls at d
3.43(3H, s), 3.35(3H, s), and 3.32(3H, s) were also determined in
the 1H NMR spectrum, which, along with the above mentioned
three secondary methyls, were compatible with three methylated
6-deoxypyranoses when examining the 13C and DEPT NMR data
which exhibited forty-two carbon signals, with eight methyls, nine
methylenes, twenty methines, and five quaternary carbons being
categorized (Tables 2 and 3). With the exception of the 13C and
DEPT NMR signals assignable to three sugars, the remaining reso-
nances were very similar to those of glaucogenin C, a known steroi-
dal aglycone isolated previously from Cynanchum atratum [7]. The
main differences were observed for glycosidation shifts at C-2
(ꢁ3.1), C-3 (+6.5), and C-4 (ꢁ4.9) in aglycone moiety of 5, so the
oligosaccharide chain was determined to link with the C-3 hydro-
xyl of 5, which was also confirmed, with the aid of HSQC spectrum
for determining the direct carbon-proton linkages, by the long-
range 1H–13C correlation of the signal of H-10 with the signal of
C-3 in the HMBC spectrum. After the anomeric protons were iden-
tified, the 1H, 1H-COSY experiment, coupled with the HSQC spec-
trum, was very effective in determining the spin systems within
the sugar moieties because of the large differences of the chemical
shifts and the relatively large coupling constants theoretically. One
b-thevetopyranose and two b-cymaropyranoses in the three sugars
were suggested by comparing the 1H and 13C NMR spectroscopic
data of 5 with those in the literature [12], which were supported
D
D
D
D
(C-3). This sequence was further substantiated by the positive ESI
mass spectrum. The ESI MSn exhibited the ion peaks: full scan
MS at m/z 831.4 [M+Na]+, MS/MS at 785.5 [M-46+Na]+, MS3 at
641.4 [M-46-cym+Na]+, and MS4 at 497.2 [M-46-cym-cym+Na]+.
Thus, compound 5 was established to be glaucogenin C 3-O-b-
D-
cymaropyranosoyl-(1 ? 4)-b- -cymaropyranosoyl-(1 ? 4)-b-D-
D
thevetopyranoside with the triglycoside being new, and was given
the trivial name of stauntoside D.
3.3. Stauntoside E (7)
The molecular formula of 7 was determined to be C49H74O19
based on the pseudomolecular ion peak at m/z 989.4727 [M+Na]+
in its positive HRESI-MS. The IR spectrum showed the absorption
bands for hydroxy (3454 cmꢁ1), carbonyl (1754, 1725 cmꢁ1), and
olefinic (1658 cmꢁ1) groups. The 1H NMR spectrum of 7 displayed
the diagnostic signals of steroidal glycoside, with a 13,14:14,15-
disecopregnane-type skeleton aglycone being exhibited typically
by two tertiary methyls and one oxymethylene (see Section 2),
and with four 6-deoxy being shown by four anomeric proton
signals at dH 4.28 (1H, d, J = 7.2 Hz, H-10), 4.75 (1H, d, J = 9.6 Hz,
H-100), 4.69 (1H, d, J = 10.2 Hz, H-1000), and 4.90 (1H, d, J = 3.6 Hz,
H-10000), which correlated to the corresponding anomeric carbon
signals at dC 100.7 (C-10), 97.8 (C-100), 99.2 (C-1000), and 99.7
(C-10000), respectively, in the HSQC spectrum, and four methyls at
dH 1.12 (12H, ov, H-60, 600, 6000, and 60000) (Table 1). In addition, four
methoxyls at d 3.43 (3H, s), 3.33 (3H, s), 3.32 (3H, s), and 3.23
(3H, s) were also determined in the 1H NMR spectrum. The four
methoxyls were compatible with the four methylated 6-deoxypyr-
anoses when examining the 13C and DEPT NMR data which exhib-
ited 49 carbon signals, with 10 methyls, nine methylenes, 24
methines, and six quaternary carbons being categorized (Tables 2

86
J.-Q. Yu et al. / Steroids 78 (2013) 79–90
Fig. 4. Observed NOE correlations in the oligosaccharide moiety of stauntoside F
(8).
Fig. 3. Observed NOE correlations in the oligosaccharide moieties of stauntosides E
(7) and G (9).
gars by three anomeric proton signals at d 4.62 (1H, d, J = 9.5 Hz,
H-10), 4.88 (1H, d, J = 9.5 Hz, H-100), and 4.81 (1H, brs, H-1000), which
correlated to the corresponding anomeric carbon signals at dC 96.9
(C-10), 97.5 (C-100), and 97.6 (C-1000), respectively, in the HSQC
spectrum, and three secondary methylic signals at d 1.13 (3H, d,
J = 6.0 Hz, H-60), 1.17 (3H, d, J = 6.0 Hz, H-600), and 1.09 (3H, d,
J = 6.5 Hz, H-6000). In addition, two methoxyls at d 3.31 (3H, s) and
3.29 (3H, s) were also determined in the 1H NMR spectrum. The
1H, 1H-COSY experiment, coupled with the HSQC spectrum, estab-
and 3). The 1H and 13C NMR resonances from the aglycone moiety
of 7 were very superimposable over those of the aglycone moiety
of stauntoside A, a known steroidal glycoside isolated previously
from C. stauntoi [8], confirming the presence of the same aglycone
between 7 and stauntoside A, and demonstrating the attachment
of a tetrasaccharide moiety to C-3 of the aglycone of 7 through oxy-
gen atom. The long-range 1H-13C correlation of the signal of H-10
with the signal of C-3 in the HMBC spectrum confirmed the con-
nection. After the anomeric protons were identified, the 1H, 1H-
COSY experiment, coupled with the HSQC spectrum, established
the spin systems within the sugar moieties. By comparing the 1H
and 13C NMR spectroscopic data of 7 with those in the literature
lished the spin systems within the sugar moiety. One b-
D-cymaro-
pyranose, one b- -digitoxopyranose, and one -cymaropyranose
D
a-L
were determined or speculated by comparing the NMR data of 8
with those in the literature [17], which were also supported by
the splitting patterns and coupling constants of the above mention
anomeric proton signals, and by the NOE correlations of H-10ax and
H-20eq, H-50ax, and OCH3-30ax, of H-100ax and H-200eq and H-500ax, of H-
[8], the four sugars were suggested to include one b-
D-thevetopyr-
anose, two b- -cymaropyranoses, and one -diginopyranose,
D
a-L
respectively, which were supported by the splitting patterns and
coupling constants of the anomeric proton signals mentioned
above, and by the NOE correlations of H-10ax and H-30ax and
H-50ax, of H-100 and H-40ax, Me-60eq, H-200eq, and H-500ax, of H-1000
1000 and H-2000eq, among others (Fig. 4), in the NOESY spectrum.
eq
Compound 8 was subjected to acid hydrolysis and GC analysis as
described above in Sections 2.4 and 2.5, which gave one
pyranose, one -cymaropyranose, and another kind of sugar units.
Because of the lack of reference substance, -cymaropyranose
D-digitoxo-
ax
ax
L
and H-400ax, Me-600eq, and H-5000ax, of H-10000 and H-4000ax, Me-6000
,
eq
eq
D
and H-20000eq, among others (Fig. 3), in the NOESY spectrum.
could not be determined in the GC test. The linkages of the three
sugars were ascertained by the HMBC spectrum, which showed
Compound 7 was subjected to acid hydrolysis and GC analysis as
the long-range 1H–13C correlations from dH 4.81(H-1000 of
a
-
L
-
described above in Sections 2.4 and 2.5, which gave one
pyranose and two other kinds of sugar units. Because of the lack of
reference substances, b- -cymaropyranose and -diginopyranose
D-theveto-
cymaropyranose) to dC 80.1 (C-400 of b-
dH 4.88 (H-100 of b- -digitoxopyranose) to dC 82.2 (C-40 of b-
cymaropyranose), and from dH 4.62 (H-10 of b-
D
-digitoxopyranose), from
D
a-L
D
D
-
units could not be determined in the GC test. The sequence of the
D
-cymaropyranose)
four sugars was confirmed by the cross peaks in the HMBC exper-
to dC 76.2 (C-3). This sequence was further substantiated by the po-
sitive ESI mass spectrum. The ESI MSn exhibited the ion peaks: full
scan MS at m/z 815.4 [M+Na]+, MS/MS at 671.3 [M-cym+Na]+, MS3
at 541.1 [M-cym-digt+Na]+, and MS4 at 397.0 [M-cym-digt-
cym+Na]+. Thus, the structure of compound 8 was determined to
iment from dH 4.90 (H-10000 of
of b-
nose) to dC 81.9 (C-400 of b-
(H-100 of b- -cymaropyranose) to dC 81.6 (C-40 of b-
nose), and from dH 4.28 (H-10 of b-
-thevetopyranose) to dC 76.8
a-L
-diginopyranose) to dC 80.9 (C-4000
D
-cymaropyranose), from dH 4.69 (H-1000 of b-
-cymaropyranose), from dH 4.75
-thevetopyra-
D-cymaropyra-
D
D
D
D
be stauntogenin 3-O-
a-
L-cymaropyranosoyl-(1 ? 4)-b-
D-digitoxo-
(C-3). This sequence was further substantiated by the positive ESI
mass spectrum. The ESI MSn exhibited the ion peaks: full scan
MS at m/z 989.6 [M+Na]+, MS/MS at 845.5 [M-dign+Na]+, MS3 at
701.3 [M-dign-cym+Na]+, and MS4 at 557.1 [M-dign-cym-
cym+Na]+; MS5 at 397.0 [M-dign-cym-cym-the+Na]+. So, the
structure of compound 7 was elucidated to be stauntogenin
pyranosoyl-(1 ? 4)-b-
D
-cymaropyranoside with the triglycoside
being new, and was given the trivial name of stauntoside F.
3.5. Stauntoside G (9)
The positive HRESI-MS of 9 gave a [M+Na]+ peak at m/z
989.4701, corresponding to the quasi-molecular formula of C49H74-
O19Na. The IR spectrum showed the absorption bands for hydroxy
3-O-
a
-
L
-diginopyranosoyl-(1 ? 4)-b-
D
-cymaropyranosoyl-(1 ?
4)-b-
D
-cymaropyranosoyl-(1 ? 4)-b-
D-thevetopyranoside with
the tetraglycoside being new, and was given the trivial name
of stauntoside E.
(3454 cmꢁ1), carbonyl (1659 cmꢁ1), and olefinic (1615 cmꢁ1
)
groups. The 1H NMR spectrum revealed the diagnostic signals of
steroidal glycoside, with a 14,15-secopregnane-type skeleton agly-
cone being exhibited by two tertiary methylic groups at d 1.10 (3H,
s, H-19) and 1.60 (3H, s, H-21), two methineoxy groups at d 3.94
(1H, m, H-3) and 4.26 (1H, m, H-16), and two oxymethylenes at
d 3.72 (1H, dd, J = 10.5, 3.5 Hz, Hb-15) and 4.31 (1H, t, J = 10.5 Hz,
3.4. Stauntoside F (8)
The molecular formula of 8 was determined to be C41H60O15 by
the pseudomolecular ion peak at m/z 815.3858 [M+Na]+ in the po-
sitive HRESI-MS. The IR spectrum showed the absorption bands for
hydroxy (3494 cmꢁ1), carbonyl (1756, 1726 cmꢁ1), and olefinic
(1660 cmꢁ1) groups. A detailed comparison between compounds
8 and 7 showed that they have the consistent 1H and 13C NMR
spectroscopic data from their aglycone moieties (see Section 2
and Table 2). With the exception of the aglyconic signals, the 1H
NMR spectrum showed the diagnostic signals of three 6-dexoysu-
H -15), and at d 3.89 (1H, d, J = 9.5 Hz, H-18a) and 5.23 (1H, d,
a
J = 9.5 Hz, H-18b). In addition, one olefinic proton signal at d 6.23
(1H, br s, H-6) was also determined in the 1H NMR spectrum.
The 13C NMR spectrum showed 49 signals. With the aid of DEPT
experiment, 28 signals were assigned to four 6-dexoysugars, and
21 to the aglycone moiety with four olefinic carbon signals
observed at d 164.9 (s), 162.3 (s), 131.0 (s), and 126.5 (d), and

J.-Q. Yu et al. / Steroids 78 (2013) 79–90
87
and L-digitoxopyranose could not be determined in the GC test.
The sequence of the sugar chain was further substantiated by the
positive ESI mass spectrum. The ESI MSn exhibited the ion peaks:
full scan MS at m/z 989.5 [M+Na]+, MS/MS at 929.5 [M-60+Na]+,
MS3 at 785.4[M-60-dign+Na]+, MS4 at 641.3[M-60-dign-cym+Na]+,
and MS5 at 497.2 [M-60-dign-cym-cym+Na]+. Therefore, the
structure of compound 9 was deduced to be stauntogenin A 3-O-
a-
L
-diginopyranosoyl-(1 ? 4)-b-
D-cymaropyranosoyl-(1 ? 4)-b-D-
cymaropyranosoyl-(1 ? 4)-b- -thevetopyranoside, and was given
D
the trivial name of stauntoside G.
Fig. 5. Key HMBC correlations in the aglycone moiety of 9.
3.6. Stauntoside H (10)
one conjugated ketonic carbonyl group observed at d 187.0. After
the directly linked carbon-proton were determined by HSQC
experiment, the long-range 1H–13C correlations in the HMBC spec-
trum from dH 1.10 (H-19) to dC 33.7 (t, C-1), 42.5 (s, C-10), 164.9 (s,
C-5), and 162.3 (s, C-9), from dH 6.23 (H-6) to dC 38.9 (t, C-4), 42.5,
and 131.0 (s, C-8), from dH 2.57 and 2.91 (H-4a, 4b) to dC 164.9 and
126.5 (d, C-6), and from dH 2.41 and 2.44 (H2-11) to dC 131.0 and
162.3 suggested the presence of a 5,8-cyclohexadien-7-one system
(Fig. 5). Considering the molecular formula, and coupled with the
13C and DEPT NMR spectroscopic data, one hydroxyl was proposed
to be linked at C-14, which was confirmed by comparing to hirun-
digoside D, a known steroidal glycoside isolated from the roots of
Vincetoxicum hirundinaria that contains a b-hydroxyl at C-14 in
the aglycone moiety [18], and by the long-range 1H–13C correla-
tions from dH 3.89 (1H, d, J = 9.5 Hz, H-18a) and 5.23 (1H, d,
J = 9.5 Hz, H-18b) to dC 105.7(C-14) in the HMBC experiment
(Fig. 5). Because of the failure of detecting the labile protons from
hydroxyls, the b orientation of 14-hydroxyl was also confirmed by
comparing the 13C NMR spectroscopic data with those of
hirundigoside D. So, the aglycone was elucidated as 3b, 14b-dihy-
droxy-14:16, 15:20, 18:20-triepoxy-14, 15-secopregnane-5,
8-dien-7-one, and was named as stauntogenin A. The 13C NMR
resonances from the sugar moiety of 9 were very superimposable
over those of the sugar moiety of 7 (see Table 3), speculatively
The positive HRESI-MS of compound 10 exhibited a quasi-
molecular ion peak at m/z 991.4845[M+Na]+, which established
the molecular formula C49H76O19. The IR spectrum showed the
absorption bands for hydroxy (3445 cmꢁ1
)
and olefinic
(1623 cmꢁ1) groups. The 1H NMR spectrum revealed the diagnostic
signals of steroidal glycoside, with a 14,15-secopregnane-type
skeleton aglycone being exhibited by two tertiary methyls at d
1.38 (3H, s, H-19) and 1.61 (3H, s, H-21), two methineoxy groups
at d 4.46 (1H, t, J = 8.0 Hz, H-3) and 5.17 (1H, m, H-16), and two
methyleneoxy groups at d 3.77 (1H, dd, J = 10.5, 4.0 Hz, Hb-15)
and 4.25 (1H, t, J = 10.5 Hz, H -15), and at d 4.06 (1H, ov, H-18a)
a
and 5.17 (1H, ov, H-18b). In addition, three olefinic proton signals
at d 6.44 (1H, d, J = 10.0 Hz, H-7), 6.12 (1H, d, J = 10.0 Hz, H-6),
and 5.68 (1H, s, H-4) were also determined in the 1H NMR spec-
trum. The 13C NMR spectrum showed 49 signals. Based on the
DEPT experiment, 28 signals were assigned to four 6-dexoysugars,
and 21 to the aglycone moiety with four olefinic carbon signals ob-
served at d 143.8 (s), 131.4 (d), 130.2 (d), and 124.3 (d). The long-
range 1H–13C correlations in the HMBC spectrum from dH 5.68 to
dC 27.2 (t, C-2), 37.4 (s, C-10), and 130.2 (d, C-6), from dH 6.12 to
dC 37.4, 75.8 (s, C-8), and 124.3 (d, C-4), from dH 6.44 to dC 55.8
(d, C-9), 111.3 (s, C-14), and 143.8 (s, C-5), and from dH 1.38 (H-
19) to dC 36.6 (t, C-1), 55.8, 37.4, and 143.8, suggested the presence
of a 4,6-diene system in the 14,15-secopregnane-type skeleton
aglycone moiety (Fig. 6). Further, considering the molecular for-
mula, and coupled with the 13C and DEPT NMR spectroscopic data,
two hydroxyls were proposed to be linked at C-8 and C-14, respec-
tively, which were confirmed by the HMBC correlations from dH
6.18 (C8-OH) to dC 55.8, 75.8, 111.3, and 131.4(d, C-7), and from
dH 8.02 (C14-OH) to dC 58.5 (s, C-13), 75.8, and 111.3. Based on
the generally accepted b-orientation of Me-19 in 14,15-secopregn-
establishing the same
a-
L
-diginopyranosoyl-(1 ? 4)-b- -cymaro-
D
pyranosoyl-(1 ? 4)-b-
D
-cymaropyranosoyl-(1 ? 4)-b-D-theveto-
pyranosoyl sugar chain connected to C-3 through oxygen atom,
which was confirmed by detailed analysis of the HMBC spectrum
(data not shown), of the 1H, 1H-COSY spectrum, and of the NOESY
experiment (Fig. 3), as well as of the acid hydrolysis and GC
analysis as described above in Sections 2.4 and 2.5, which gave
one
D-thevetopyranose and another two kinds of sugar units.
Because of the lack of reference substances,
D
-cymaropyranose
ane-type skeleton, the b-orientation of the C14-OH and the a-ori-
O
O
OH
H
H
O
OH
RO
10 R = Rh
19
O
H
C
3
19
O
C10
C7
H
C14
C
11
O
19
14
HO
H
9
8
H
O
14
9
H
OH
H
O
H
8
RO
10 R = Rh
Fig. 6. Key HMBC correlations and observed NOE correlations in the aglycone moiety of 10.

88
J.-Q. Yu et al. / Steroids 78 (2013) 79–90
entation of C8-OH were determined by the different nuclear Over-
hauser effect (NOE) spectra, i.e. irradiation of the signal of Me-19
showed NOE enhancement of the signal of C14-OH, and irradiation
of the signal of H-9 showed NOE enhancement of the signal of C8-
trum of 13 revealed the diagnostic signals of steroidal glycoside,
with a 14,15-secopregnane-type skeleton aglycone being exhibited
typically by two tertiary methylic groups and one oxymethylene
(see Section 2), and with three 6-deoxysugars being shown by
three anomeric proton signals at d 4.32 (1H, d, J = 7.5 Hz, H-10),
4.90 (1H, d, J = 9.3 Hz, H-100), and 4.68 (1H, d, J = 9.9 Hz, H-1000),
which correlated to the corresponding anomeric carbon signals at
dC 100.7 (C-10), 98.6 (C-100), and 99.5 (C-1000), respectively, in the
HSQC spectrum, and three methyls at d 1.28 (6H, ov, H-60 and
6000) and 1.24 (3H, d, J = 6.0 Hz, H-600) (Table 1). In addition, three
methoxyls at d 3.43 (6H, ov) and 3.62 (3H, s) were also determined
in the 1H NMR spectrum, which were compatible with three meth-
ylated 6-deoxypyranoses when examining the 13C and DEPT NMR
data which exhibited forty-one carbon signals, with eight methyls,
10 methylenes, 18 methines, and six quaternary carbons being cat-
egorized (Tables 2 and 3). With the exception of the 13C and DEPT
NMR signals assignable to three sugars, the remaining resonances
were very similar to those of anhydrohirundigenin, a known steroi-
dal aglycone isolated previously from Cynanchum hancockianum
[19]. The main differences were observed for glycosidation shifts
at C-2 (ꢁ2.6), C-3 (+6.5), and C-4 (ꢁ3.8) in aglycone moiety of
13, thus the oligosaccharide chain was determined to be linking
at the C-3 hydroxyl of 13, which was also confirmed, with the
aid of HSQC spectrum for determining the direct carbon-proton
linkages, by the long range 1H–13C correlation of the signal of H-
10 with the signal of C-3 in the HMBC spectrum. After the anomeric
protons were identified, the 1H, 1H-COSY experiment, coupled with
the HSQC spectrum, established the spin systems within the sugar
OH (Fig. 6). So, the aglycone was elucidated as 3b,8a,14b-trihy-
droxy-14:16,15:20,18:20-triepoxy-14,15-secopregnane-4,6-diene,
and was named as stauntogenin B. With the exception of the agly-
conic signals, the 1H NMR spectrum of 10 also showed four second-
ary methylic signals at dH 1.37 (6H, m, H-600, 6000), 1.47 (3H, d,
J = 5.0 Hz, H-60), and 1.56 (3H, d, J = 7.5 Hz, H-60000), and four ano-
meric proton signals at dH 5.31 (1H, br d, J = 9.0 Hz, H-100), 5.08
(1H, br d, J = 9.0 Hz, H-1000), 5.22 (1H, br d, J = 3.5 Hz, H-10000), and
4.78 (1H, d, J = 7.5 Hz, H-10). These anomeric proton signals corre-
lated to their corresponding anomeric carbon signals at dC 103.2
(C-10), 98.8 (C-100), 100.2 (C-1000), and 101.1 (C-10000), respectively,
in the HSQC spectrum. The 1H, 1H-COSY experiment, coupled with
the HSQC spectrum, established the spin systems within the sugar
moiety. These data indicated the presence of four 6-deoxysugars,
with three being b-linkages and one being
a-linkage. Comparing
the 13C NMR data of the sugars with those of 9 and those in the lit-
erature [17] suggested that 10 possessed a similar sugar chain with
9 except that the terminal
a-L-diginopyranose in 9 was replaced by
a a-L-cymaropyranose in 10. This conclusion was confirmed by the
cross peaks in the HMBC spectrum from dH 5.22 (H-10000 of
cymaropyranose) to dC 82.2 (C-4000 of b-
dH 5.08 (H-1000 of b- -cymaropyranose) to dC 83.3 (C-400 of b-
cymaropyranose), from dH 5.31 (H-100 of b-
dC 82.7 (C-40 of b-
b- -thevetopyranose) to dC 75.5 (C-3), and by the NOE correlations
a
-L
-
D-cymaropyranose), from
D
D
-
D
-cymaropyranose) to
D
-thevetopyranose), and from dH 4.78 (H-10 of
D
moiety. The b-D-thevetopyranose, b-D-cymaropyranose, and b-L-
of H-10ax and H-30ax and H-50ax, of H-100 and H-40ax, Me-60eq
,
diginopyranose were speculated by comparing the 1H and 13C
NMR spectroscopic data of 13 with those in the literature [20],
and with those of the corresponding sugars of cynanversicoside
G [20], cynanversicoside A [21], and compound 5. These sugars
were also supported by the splitting patterns of the above men-
tioned anomeric proton signals. Compound 13 was subjected to
acid hydrolysis and GC analysis as described above in Sections
ax
H-200eq, and H-500ax, of H-1000 and H-400ax, Me-600eq, and H-5000ax, of
ax
H-10000 and H-4000ax, Me-6000eq, and H-20000eq, among others (Fig. 7),
eq
in the NOESY spectrum. This sequence was further substantiated
by the positive ESI mass spectrum. The ESI MSn exhibited the ion
peaks: full scan MS at m/z 991.5 [M+Na]+, MS/MS at 829.5 [M-
18-cym+Na]+, MS3 at 685.3 [M-18-cym-cym+Na]+, MS4 at 541.2
[M-18-cym-cym-cym+Na]+, and MS5 at 381.2 [M-18-cym-cym-
cym-the+Na]+.Compound 10 was subjected to acid hydrolysis
and GC analysis as described above in 2.4 and 2.5, which gave
2.4 and 2.5, which gave one
kinds of sugar units. Because of the lack of reference substances,
-cymaropyranose and -diginopyranose could not be determined
D-thevetopyranose and two other
D
L
one
D
-thevetopyranose, one
L
-cymaropyranose, and another kind
-cymar-
in the GC test. The linkages of the three sugars were ascertained
of sugar unit. Because of the lack of reference substance,
opyranose could not be determined in the GC test. On the basis of
the above findings, the structure of 10 was assigned as stauntoge-
nin B 3-O-
(1 ? 4)-b-
D
by the HMBC spectrum, which showed long range 1H–13C correla-
tions from dH 4.68 (H-1000 of b-
b-
to dC 81.6 (C-40 of b-
b-D-thevetopyranose) to dC 78.1 (C-3). This sequence was further
L
-diginopyranose) to dC 82.4 (C-400 of
D
-cymaropyranose), from dH 4.90 (H-100 of b-
-thevetopyranose), and from 4.32 (H-1’ of
D-cymaropyranose)
a-L
-cymaropyranosoyl-(1 ? 4)-b-
D
-cymaropyranosoyl-
D
D
-cymaropyranosoyl-(1 ? 4)-b-
D
-thevetopyranoside
with the tetraglycoside being new, and was given the trivial the
name of stauntoside H.
substantiated by the positive ESI mass spectrum. The ESI-MSn
exhibited the ion peaks: full scan MS at m/z 815.5 [M+Na]+, MS/
MS at 671.3 [M-dign+Na]+ and MS3 at 527.2 [M-dign-cym+Na]+.
Thus, the structure of 13 was determined to be anhydrohirundige-
3.7. Stauntoside I (13)
nin
3-O-b-L-diginopyranosoyl-(1 ? 4)-b-D-cymaropyranosoyl-
(1 ? 4)-b-
D
-thevetopyranoside with the triglycoside being new,
The molecular formula of 13 was determined to be C42H64O14
based on its positive HRESI-MS data at m/z 815.4188[M+Na]+.
The IR spectrum showed the absorption bands for hydroxy
(3451 cmꢁ1) and olefinic (1681 cmꢁ1) groups. The 1H NMR spec-
and was given the trivial name of stauntoside H.
3.8. Stauntoside J (14)
The positive HRESI-MS of 14 exhibited a quasi-molecular ion
peak at m/z 799.3892 [M+Na]+, corresponding to the molecular for-
mula C41H60O14. The IR spectrum showed the absorption bands for
hydroxy (3485 cmꢁ1) and olefinic (1677 cmꢁ1) groups. A detailed
comparison between compounds 14 and 2 indicated that they have
the consistent 1H and 13C NMR spectroscopic data from their agly-
cone moieties (see Section 2 and Table 2),, which was confirmed to
be deoxyamplexicogenin A by detailed analysis of 2D NMR spectra
(data not shown). With the exception of the aglycone signals, the
1H NMR spectrum of 14 revealed the diagnostic signals of three
6-deoxysugars by three anomeric proton signals at d 4.32 (1H, d,
Fig. 7. Observed NOE correlations in the oligosaccharide moiety of stauntoside H
(10).

J.-Q. Yu et al. / Steroids 78 (2013) 79–90
89
J = 7.5 Hz, H-10), 4.70 (1H, dd, J = 10.0, 2.0 Hz, H-1000), and 4.85 (1H,
d, J = 8.0 Hz, H-100), which correlated to the corresponding anomer-
ic carbon signals at dC 101.8 (C-10), 99.0 (C-1000), and 97.9 (C-100),
respectively, in the HSQC spectrum, and three secondary methylic
signals at d 1.11 (3H, d, J = 6.5 Hz, H-6000), 1.12 (3H, d, J = 6.5 Hz, H-
600), and 1.14 (3H, d, J = 6.0 Hz, H-60). The 1H, 1H-COSY experiment,
coupled with the HSQC spectrum, established the spin systems
within the sugar moiety. By comparing the 1H and 13C NMR
spectroscopic data of 14 with those of compounds 5 and 8, the
fields of pharmaceutics and chemotaxonomy. More than 200 C-
21 steroidal glycosides have been isolated from Cynanchum species
up to the year of 2007 [14,22,23] and further findings are continu-
ously claimed in the everyday practice of phytochemistry [23,24].
From a phytochemical point of view, it is of interest to note that
only the steroidal saponins with aglycones assignable to the aber-
rant 13,14:14,15-disecopregnane-type skeleton or the equally
abnormal 14,15-secopregnane-type skeleton were found in C. sta-
untonii, compared to six skeletal subtypes being grouped and iso-
lated in total from Cynanchum species. This is additional evidence
for the botanical classification of C. stauntonii, and they might be
important chemical markers of the species.
structures of three sugars were suggested, i.e., one b-D-thevetopyr-
anose, one b- -digitoxopyranose, and one b- -cymaropyranose,
D
D
which were confirmed by the splitting patterns of the above men-
tioned anomeric proton signals. Compound 14 was subjected to
acid hydrolysis and GC analysis as described above in Sections
Acknowledgements
2.4 and 2.5, which gave one
anose, and another kind of sugar unit. Because of the lack of refer-
ence substance, -cymaropyranose unit could not be determined in
D-thevetopyranose, one D-digitoxopyr-
We thank associate Professor L Ma for the sample authentica-
tion. This work was supported by grants from National Science
and Technology Project of China (2011ZX09307-002-01).
D
the GC test. The linkages of the three sugars were confirmed by the
HMBC spectrum, which showed long range 1H–13C correlations
from dH 4.70 (H-1000 of b-
b-
nose) to dC 81.9 (C-40 of b-
(H-10 of b-
-thevetopyranose) to dC 74.6 (C-3). This sequence was
D
-cymaropyranose) to dC 81.6 (C-400 of
Appendix A. Supplementary data
D
-digitoxopyranose), from dH 4.85 (H-100 of b-
-thevetopyranose), and from dH 4.32
D-digitoxopyra-
D
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2012.10.
007.
D
further substantiated by the positive ESI mass spectrum. The ESI-
MSn exhibited the ion peaks: full scan MS at m/z 799.5 [M+Na]+,
MS/MS at 655.3 [M-cym+Na]+, and MS3 at 525.2 [M-cym-
digt+Na]+. Hence, the structure of compound 14 was elucidated
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