Steroidal glycosides from the roots of Cynanchum stauntonii and their effects on the expression of iNOS and COX-2

Article abstract of DOI:10.1016/j.phytol.2016.02.016

Six new steroidal glycosides, named stauntosides O-T (1-6), along with eight known compounds (7-14), were obtained from the 95% aqueous ethanol extract of the roots of Cynanchum stauntonii. Their chemical structures were elucidated by IR, HR ESI-MS/MS, ¹H- and ¹³C NMR, ¹H-¹H COSY, HSQC, HSQC-TOCSY, and HMBC spectroscopic analyses, which showed interesting 13,14:14,15-disecopregnane-type or 14,15-secopregnane-type C₂₁ steroidal glycosides. The glycosides' anti-inflammatory effects were investigated by detecting the inhibitory effects of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) on RAW246.7 murine macrophage cells stimulated by lipopolysaccharide (LPS). Our results showed that compounds 1, 5, 8, 9, 11, and 13 could significantly inhibit iNOS expression, and compounds 5 and 7 could clearly reduce COX-2 expression in RAW246.7 cells stimulated by LPS compared to cells stimulated with LPS and not treated with other compounds. Thus, compounds 1, 5, 7-9, 11, and 13 have the potential to mediate anti-inflammatory effects, with compound 5 having a greater anti-inflammatory effect than the other compounds.

Full text of DOI:10.1016/j.phytol.2016.02.016

Phytochemistry Letters 16 (2016) 38–46
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Short communication
Steroidal glycosides from the roots of Cynanchum stauntonii and their
effects on the expression of iNOS and COX-2
Chang-Zhi Laia,1, Jian-Xin Liub,c,1, Shu-Wen Panga, Yi Daia, Hua Zhoub, Zhen-Qiang Mud,
b
a,
Jun Wud, Jin-Shan Tanga, , Liang Liu , Xin-Sheng Yao
*
*
a
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China
State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Avenida Wailong, Taipa, Macau, People’s
b
Republic of China
c
College of Pharmacy, Hunan University of Medicine, Huaihua 418000, People’s Republic of China
Guangzhou Xiangxue Pharmaceutical Ltd. Co., Guangzhou 510663, People’s Republic of China
d
A R T I C L E I N F O
A B S T R A C T
Article history:
Six new steroidal glycosides, named stauntosides O-T (1–6), along with eight known compounds (7–14),
were obtained from the 95% aqueous ethanol extract of the roots of Cynanchum stauntonii. Their chemical
structures were elucidated by IR, HR ESI-MS/MS, 1H- and 13C NMR, 1H-1H COSY, HSQC, HSQC-TOCSY, and
HMBC spectroscopic analyses, which showed interesting 13,14:14,15-disecopregnane-type or 14,15-
secopregnane-type C21 steroidal glycosides. The glycosides’ anti-inflammatory effects were investigated
by detecting the inhibitory effects of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-
2) on RAW246.7 murine macrophage cells stimulated by lipopolysaccharide (LPS). Our results showed
that compounds 1, 5, 8, 9, 11, and 13 could significantly inhibit iNOS expression, and compounds 5 and 7
could clearly reduce COX-2 expression in RAW246.7 cells stimulated by LPS compared to cells stimulated
with LPS and not treated with other compounds. Thus, compounds 1, 5, 7–9, 11, and 13 have the potential
to mediate anti-inflammatory effects, with compound 5 having a greater anti-inflammatory effect than
the other compounds.
Received 28 December 2015
Received in revised form 14 February 2016
Accepted 26 February 2016
Available online xxx
Keywords:
Cynanchum stauntonii
C21 steroidal glycoside
Chemical constituents
Anti-inflammatory activity
ã 2016 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
in vitro also indicated that seco-pregnane steroids from C.
stauntonii and C. glaucescens exhibited inhibitory activity against
The dried roots of Cynanchum stauntonii (Decne.) Schltr.ex Lévl.,
together with the other species of the same genus, C. glaucescens
(Decne.) Hand.-Mazz., are recorded in the Chinese Pharmacopoeia
(Commission, 2010) as “Bai-Qian” and have been used as
antitussive and expectorant in China for a long time. Both of
them belong to the family Asclepiadaceae. C. stauntonii is widely
distributed in the south-central region of China. Chemical
investigations revealed that the roots primarily contained C21
steroidal glycosides, especially with the aberrant 13,14:14,15-
disecopregnane-type or 14,15-secopregnane-type skeleton. It is
well known that C21 steroidal constituents exhibit considerable
bioactivities, including hypolipidemic, antitumor, and anti-inflam-
matory activities (Day et al., 2001; Zhu et al., 1999). Recent studies
Na+/K+-ATPase (Shibano et al., 2012) and selective inhibition to
alphavirus-like RNA viruses (Li et al., 2007). In our search for anti-
inflammatory constituents from the plant of C. stauntonii, six new
C21 steroidal glycosides (stauntosides O-T, 1–6), along with eight
known compounds (7–14), were obtained from the dried roots of
C. stauntonii. In this study, we describe the isolation and structural
elucidation of these new compounds, as well as the anti-
inflammatory activity of C21 steroidal glycosides.
2. Experimental
2.1. General methods
Optical rotations were obtained on a P-1020 digital polarimeter
(Jasco Corporation, Tokyo, Japan). IR spectra were recorded on a
Jasco FTIR-480 Plus spectrometer. NMR spectra were measured
with Bruker AV 300, 400, 500 and 600 NMR instruments. Chemical
* Corresponding authors.
E-mail addresses: gztangjinshan@126.com (J.-S. Tang), tyaoxs@jnu.edu.cn
shifts were given in ppm (
d) relative to chemical shifts of solvent
(X.-S. Yao).
1
resonances (C5D5N: 8.74 and 150.3 ppm). HR ESI-MS spectra were
Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.phytol.2016.02.016
1874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

C.-Z. Lai et al. / Phytochemistry Letters 16 (2016) 38–46
39
Table 1
1H and 13C NMR data of compounds 1–6 (
d in ppm, J values in Hz).
No.
1
2
3
4
5
6
dC
dH
dC
dH
dC
dH
dC
dH
dC
dH
dC
dH
1
2
37.0 (t) 1.84,m/0.97,m 37.0
1.85,m/0.97, 36.9
m
1.87,m/0.98, 37.0
m
2.12,m/1.71, 30.5
m
1.84,m/0.96,m 37.0
1.86,m/0.97, 38.0
m
2.13,m/1.72, 30.7
m
1.78,m/1.11,m
30.6 (t) 2.11,m/1.71,m 30.6
2.11,m/1.71,
m
30.5
2.12,m/1.71,m
3.83,m
30.5
78.5
1.73,m/1.39,m
3
4
78.0 (d) 3.79,m
39.5 (t) 2.58,m/2.32,
m
78.1
39.5
3.77,m
78.7
3.85,m
78.7
3.82,m
78.7
3.84,m
2.64,m/2.44,m
2.56,m/2.29, 39.4
m
2.65,m/2.34, 39.5
m
2.65,m/2.33,m 39.5
2.63,m/2.32, 39.7
m
5
6
141.1 (s)
121.0
(d)
141.1
121.0
141.1
120.8
141.1
120.9
141.1
120.9
140.7
122.2
5.43, d(5.4)
5.42,d(4.9)
5.35,d(5.2)
5.35,d(5.2)
5.36,d(4.8)
5.36,overlapped
2.50,m/2.22,m
7
29.0 (t) 2.66,m/2.17,m 28.9
2.66,m/2.16, 28.9
m
2.62,m/2.12, 28.9
m
2.62,m/2.12,m 28.9
2.63,m/2.10, 26.0
m
8
9
10
11
41.2 (d) 2.52,m
53.7 (d) 1.25,t(10.4)
39.2 (s)
24.5 (t) 2.64,m/1.40,
m
41.2
53.7
39.2
24.4
2.52,m
1.25,m
41.1
53.7
39.1
2.48,m
1.24,t(10.4)
41.2
53.7
39.2
2.47,m
1.23,t(10.3)
41.2
53.7
39.2
2.47,m
1.24,t(10.5)
31.8
46.3
37.4
1.96,m
1.65,m
2.62,m/1.39, 24.4
m
2.04,m/1.39, 30.4
m
2.62,m/1.39, 24.4
m
2.06,m/1.41, 30.5
m
2.62,m/1.38,m 24.4
2.04,m/1.39,m 30.5
114.9
2.62,m/1.38, 20.5
m
2.06,m/1.38, 30.8
m
1.48,m/1.41,m
12
13
14
15
30.5 (t) 2.06,m/1.41,m 30.5
2.11,dd(12.2,4.3)/
1.60,m
114.9
(s)
176.0
(s)
114.9
176.0
68.3
114.8
114.9
49.2
175.9
176.0
176.0
109.5
68.3 (t) 4.28,m/3.98,
m
4.28,m/3.96, 68.2
m
4.25,m/3.97, 68.3
m
4.28,m/3.96,m 68.3
4.26,m/3.94, 71.8
m
4.05,d(9.3)/3.83,m
16
17
18
76.1 (d) 5.48,m
56.7 (d) 3.41,m
76.0
56.7
144.4
5.47,m
3.58,m
6.51,s
76.0
56.7
144.3
5.46,m
3.57,m
6.49,s
76.1
56.7
144.4
5.46,m
3.57,d(8.4)
6.51,s
76.0
56.7
144.4
5.46,q(7.9)
3.57,m
6.50,s
77.6
58.5
16.0
4.47,m
2.32,d(2.6)
1.10,s
144.4
(d)
6.52,s
19
20
18.4 (q) 0.86,s
119.0
(s)
18.4
119.0
0.87,s
18.3
119.0
0.81,s
1.56,s
18.3
119.0
0.80,s
1.57,s
18.3
119.0
0.81,s
1.57,s
19.7
114.1
0.93,s
1.74,s
21
10
25.3 (q) 1.57,s
25.3
1.57,s
25.2
25.3
25.3
24.4
b
-D-ole 4.84,dd(9.7,
b-D
-
4.90,d(9.9)
b-D
-the 4.83,d(7.6)
b
-D-the 4.84,d(7.8)
b-D
-the 4.83,d(7.7)
b-D-the 4.83,d(7.7)
98.6 (d) 1.8)
can
102.8
102.8
102.8
102.7
98.7
40.6
20
38.5(t)
2.45,m/1.83,
m
2.54,m/1.99, 75.1
m
3.94,m
75.1
3.95,m
75.1
3.92,m
75.1
3.93,m
30
79.6 (d) 3.63,m
83.7 (d) 3.61,m
72.3 (d) 3.60,m
19.4 (q) 1.50,d(5.6)
58.0 (q) 3.59,s
70.6
89.0
71.4
18.6
3.98,m
86.2
83.1
72.0
18.8
60.9
3.71,m
3.71,m
3.71,m
1.49,d(4.0)
3.95,s
86.3
83.4
72.1
19.2
61.0
3.72,m
3.73,m
3.70,m
1.48,d(6.0)
3.95,s
86.3
83.2
72.1
19.2
61.0
3.68,m
3.69,m
3.70,m
1.48,d(3.0)
3.94,s
86.4
83.3
72.0
19.2
61.0
3.69,m
3.69,m
3.70,m
1.47,d(3.5)
3.95,s
40
3.34,t(8.8)
3.56,m
50
60
1.39,d(6.0)
30-
OCH3
100
200
b
digt
-
D
-
5.58,dd(9.7,
1.8)
b
digt
100.3
38.7
-
D
-
5.29,d(9.5)
b
cym
99.2
-
D
-
5.33,d(7.1)
b
digt
99.4
-
D
-
5.53,dd(9.6,1.2)
b
cym
100.4
-
D
-
5.31,d(9.5)
b
cym
99.3
-
D
-
5.32,dd(9.8,1.5)
2.37,m/1.87,m
99.4 (d)
40.3 (t) 2.47,m/2.02,
m
69.3 (d) 4.47,m
74.6 (d) 3.63,m
71.1 (d) 4.31,m
19.6 (q) 1.61,d(6.2)
2.46,m/2.00, 37.5
m
4.50,d(2.7)
3.43,m
4.20,m
1.33,d(6.1)
2.36,m/1.87, 39.6
m
2.45,m/2.02,m 37.5
2.35,m/1.86, 37.5
m
4.22,m
3.49,m
4.19,m
300
68.0
81.3
69.8
18.5
78.5
83.7
69.7
19.0
59.3
4.09,m
3.49,m
4.24,m
1.38,d(5.8)
3.69,s
68.2
83.6
69.3
19.0
4.65,d(2.6)
3.50,m
4.31,m
78.7
83.7
69.7
18.9
59.4
78.5
83.7
69.7
19.1
59.3
4.10,m
400
3.50,m
4.23,m
1.40,d(6.2)
3.63,s
500
600
1.43,d(6.0)
1.37,d(6.2)
3.63,s
300-
OCH3
1000
a
-
L
-
5.04,br s
b
cym
100.6
-
D
-
5.10,d(9.5)
b
cym
100.1
-
D
-
5.14,dd(9.6,1.2)
b
cym
100.7
-
D
-
5.08,d(9.6)
b
cym
100.7
-
D
-
5.10,dd(9.7,1.6)
2.44,m/1.75,m
cym
98.9
32.8
2000
2.33,m/1.81, 35.6
m
3.94,m
3.99,m
2.43,m/1.75, 35.3
m
3.97,m
3.47,m
2.42,m/1.69,m 35.5
2.41,m/1.72, 35.6
m
3.92,m
3.45,m
3000
4000
74.0
78.5
77.9
82.6
77.9
82.8
3.90,d(2.4)
3.40,dd
78.0
82.8
78.0
82.7
3.96,m
3.47,m
(9.5,2.4)
4.22,m
1.31,d(6.0)
3.54,s
5000
6000
300-
66.8
18.6
57.4
4.62,m
1.37,d(6.6)
3.45,s
69.7
19.1
57.7
4.24,m
1.40,d(6.1)
3.55,s
69.8
19.0
57.8
69.7
19.1
57.8
4.19,m
1.39,d(6.4)
3.54,s
69.7
18.9
57.8
4.23,m
1.39,d(6.2)
3.55,s
OCH3
10000
b-D
-
5.02,d(7.3)
a-L
-
5.24,d(3.1)
a
-L-
5.20,d(3.2)
a-L
-
5.22,d(2.4)
a-L
-
5.24,d(3.0)
glu
103.0
dign
101.5
dign
101.6
dign
101.6
dign
101.6

40
C.-Z. Lai et al. / Phytochemistry Letters 16 (2016) 38–46
obtained with a Micromass Q-TOF mass spectrometer. HPLC was
m) and a
HPLC system equipped with a Waters e2695 system, a Waters
2998 PDA detector and an Alltech 3300 ELSD detector. Semi-
preparative HPLC was performed on a HPLC system equipped with
a Waters 1515 pump and a Waters 2489 detector, using a Sunfire
(
w
4.0 ꢀ 30.0 cm) using a CH3OH:H2O gradient to afford 13 sub-
performed with a Sunfire C18 column (4.6 ꢀ 250 mm, 5
m
fractions (D10-1ꢁD10-13). Subfraction D10-6 was subjected to
ODS CC using a gradient of CH3OH:H2O as the eluent to yield
8
subfractions (D10-6-1ꢁD10-6-8). Subfraction D10-6-4 was
subjected to Sephadex LH-20CC (CH3OH) and then applied to a
semi-preparative RP-HPLC system (CH3O H-H2O, 70: 30) to afford
compound 11 (10.0 mg). Subfraction D10-7 was separated by ODS
CC using gradient solvents of CH3OH:H2O to yield 7 subfractions
C18 column (10.0 ꢀ 250 mm, 5
(CC) was performed using silica gel (200–300 mesh, Qingdao
Haiyang Chemical Group Corp., Qingdao), ODS column (50 m,
mm). Open column chromatography
m
(D10-7-1ꢁD10-7-7). Subfraction D10-7-6
was subjected to
YMC Co., Ltd., Kyoto, Japan), and Sephadex LH-20 (Pharmacia Corp.,
United States). TLC analysis was performed on pre-coated silica gel
GF254 plates (Qingdao Haiyang Chemical Group Corp., Qingdao).
Lipopolysaccharide (LPS) used for stimulation of RAW246.7 cells
and the drug dexamethasone, used as a positive control or
standard, were obtained from Sigma Chemical Co. (St. Louis, MO,
USA).
Sephadex LH-20CC (CH3OH) and then applied to a semi-prepara-
tive RP-HPLC system (CH3OH:H2O, 70:30) to yield compounds 6
(34.0 mg), 8 (12.0 mg), and 12 (11.0 mg). Subfraction D10-7-7 was
subjected to Sephadex LH-20CC (CH3OH) and then semi-prepara-
tive RP-HPLC (CH3O H-H2O, 70: 30) to yield compounds 1 (23.0 mg)
and 9 (13.0 mg). Subfraction D10-8 was chromatographed over
silica gel CC using petroleum ether:EtOAc (7:3, 1:1, 0:100, v/v) as
the eluent to give 10 subfrations (D10-8-1ꢁD10-8-10). Subfraction
D10-8-6 was applied to a semi-preparative RP-HPLC system
(CH3OH:H2O, 70:30) to yield compound 10 (58.0 mg). Subfraction
D10-8-7 was applied to semipreparative RP-HPLC system (CH3OH:
H2O, 70:30) to yield compound 14 (86.0 mg). Subfraction D10-8-
8 was applied to a semi-preparative RP-HPLC system (CH3OH:H2O,
70:30) to yield compounds 3 (84.0 mg) and 13 (8.0 mg). Subfraction
2.2. Plant material
The Chinese Medicine “Bai-Qian,” the dried roots of C.
stauntonii, was obtained from Guangzhou Xiangxue Pharmaceuti-
cal Co., Ltd., in November, 2012. It was identified by Associate Prof.
Ying Zhang, College of Pharmacy, Jinan University. A voucher
specimen (CS201211-1) was deposited into the Institute of
Traditional Chinese Medicine and Natural Products, Jinan Univer-
sity.
D12 (39.0 g) was subjected to ODS CC (
w
4.0 ꢀ 30.0 cm) using a
gradient of CH3OH:H2O to afford 13 subfractions (D12-1ꢁD12-13).
Subfraction D12-5 was applied to a semi-preparative RP-HPLC
system (CH3OH:H2O, 70:30) to obtain compound 4 (50.0 mg).
Subfraction D12-8 was subjected to Sephadex LH-20CC (CH3OH)
and then applied to a semi-preparative RP-HPLC system (CH3OH:
H2O, 70:30) to yield compounds 2 (25.0 mg), 5 (86.0 mg), and 7
(25.0 mg).
2.3. Extraction and isolation
The dried and chopped roots (9.5 kg) of C. stauntonii were
refluxed three times in 95% (v/v) aqueous EtOH (90 L ꢀ 3). The
combined ethanol extract was evaporated under vacuum to yield a
dark-brown residue (ca. 1100 g). The residue was directly
chromatographed over an open macroporous Diaion HP 20 col-
2.3.1. Stauntoside O (1)
White amorphous powder; ½a D28
ꢃ42.5 (c 0.5, CH3OH); IR (KBr)
ꢂ
nmax: 3443, 2934, 1735, 1516 and 1068 cmꢃ1; ESI–MS (positive
mode) m/z 657.9 [M + Na]+. HR ESI–MS (positive mode) m/z
657.3260 [M + Na]+ (calcd for C34H50O11Na, 657.3251); 1H
(500 MHz, C5D5N) and 13C NMR (125 MHz, C5D5N) data see Table 1.
umns (
w
12.0 ꢀ 63.0 cm) and eluted with a gradient of EtOH:H2O
(0, 30, 50, and 95% of EtOH) to yield 4 fractions (A-D). Fraction D
(93.0 g, 95% ethanol) was applied to a silica gel column for
chromatography (
w
9.5 ꢀ 68.0 cm) using a gradient of mixtures of
petroleum ether:ethyl acetate (EtOAc) (100:0, 98:2, 95:5, 9:1, 7:3,
0:100) and EtOAc:methanol (CH3OH) (95:5, 9:1, 7:3, 0:100) to
yield 13 subfractions (D1-D13) according to their TLC profiles and
HPLC behavior. Fraction D10 (20.8 g) was subjected to open ODS CC
2.3.2. Stauntoside P (2)
White amorphous powder; ½a D28
ꢃ32.9 (c 0.5, CH3OH); IR (KBr)
ꢂ
nmax: 3447, 2938, 1739, 1515 and 1077 cmꢃ1; ESI-MS (positive
Table 1 (Continued)
No.
1
2
3
4
5
6
dC
dH
dC
dH
dC
dH
dC
dH
dC
dH
dC
dH
20000
75.8
4.01,m
31.4
2.40,m/2.09, 32.7
m
2.36,m/2.05,m 32.7
2.35,m/2.06, 31.4
m
2.41,m/2.11,m
30000
40000
50000
60000
79.2
72.2
78.9
63.4
3.99,m
4.22,m
4.25,m
76.2
68.1
68.2
3.88,m
4.09,m
4.34,m
1.59,d(6.5)
75.6
74.8
68.6
18.4
3.87,m
4.28,m
4.28,m
1.71,d(6.4)
75.6
74.8
68.5
18.4
4.01,m
4.26,m
4.29,m
1.71,d(6.3)
76.3
68.1
68.2
18.3
3.89,m
4.10,m
4.34,m
1.59,d(6.5)
4.58,m/4.38, 18.1
m
300-
55.4
3.34,s
56.2
3.54,s
56.2
3.53,s
55.5
3.35,s
OCH3
100000
b-D
-glu 4.99,d(7.8)
b-D-glu 4.97,d(7.7)
105.9
75.7
79.1
72.3
78.8
63.5
105.9
75.7
79.1
72.2
78.8
63.5
200000
300000
400000
500000
600000
4.04,t(7.5)
4.24,d(8.9)
4.19,m
3.96,m
4.55,dd
3.88,m
4.19,m
4.17,m
4.20,m
4.52,m/4.34,
m
(11.4,2.3)/
4.36,dd
(11.4,5.4)
Multiplicities by DEPT experiments in parentheses; s:quanternary, d: CH, t:CH2, and q: CH3C-atoms.

C.-Z. Lai et al. / Phytochemistry Letters 16 (2016) 38–46
41
mode) m/z 949.9 [M + Na]+. HR ESI-MS (positive mode) m/z
949.4405 [M + Na]+ (calcd for C46H70O19Na, 949.4409); 1H
(400 MHz, C5D5N) and 13C NMR (75 MHz, C5D5N) data see Table 1.
those of the standards, the glucoses in compounds 2, 4, and 5 were
determined to be d-configuration.
2.4.2. Mild acid hydrolysis of compounds 1, 3, and 6 and determination
of the absolute configuration of sugars
2.3.3. Stauntoside Q (3)
White amorphous powder; ½a D28
ꢃ45.3 (c 0.5, CH3OH); IR (KBr)
ꢂ
Each solution of compounds 1, 3, and 6 (20 mg) in CH3OH (2 mL)
was mixed with 0.05 N HCl (3 mL), stirred for 15 min at 70 ꢄC and
then evaporated in vacuum to remove CH3OH. The aqueous
solution was extracted with CHCl3 (3 mL ꢀ 3). The aqueous layer
was neutralized with Ba(OH)2 saturated with H2O. The precipitates
were filtered out and the solution dried to yield a crude sugar
fraction. The crude sugar fraction was subjected to silica gel CC,
successive eluting with CHCl3:MeOH at the ratios indicated to yield
monosugars. For compound 1, oleandrose (97:3, v/v) and
digitoxose (93:7, v/v) were obtained from the aqueous layer; for
compound 3, diginose (98:2, v/v), cymarose (97:3, v/v), and
thevetose (85:15, v/v) were obtained from the aqueous layer; and
for compound 6, diginose (98:2, v/v), cymarose (97:3, v/v), and
thevetose (85:15, v/v) were obtained from the aqueous layer.
nmax: 3495, 2935, 1734, 1450 and 1072 cmꢃ1; ESI-MS (positive
mode) m/z 976.1 [M + Na]+. HR ESI-MS (positive mode) m/z
975.4924 [M + Na]+ (calcd for C49H76O18Na, 975.4929); 1H
(400 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) data see Table 1.
2.3.4. Stauntoside R (4)
White amorphous powder; ½a D28
ꢃ31.8 (c 0.5, CH3OH); IR (KBr)
ꢂ
nmax: 3450, 2943, 1733, 1522 and 1076 cmꢃ1; ESI-MS (positive
mode) m/z 1123.9 [M + Na]+. HR ESI-MS (positive mode) m/z
1123.5300 [M + Na]+ (calcd for C54H84O23Na, 1123.5301); 1H
(600 MHz, C5D5N) and 13C NMR (75 MHz, C5D5N) data see Table 1.
2.3.5. Stauntoside S (5)
D
-digitoxose was obtained as a colorless gum with the following
White amorphous powder; [
a
]28 D ꢃ24.5 (c 0.5, CH3OH); IR
analytical properties: ½a D27
ꢂ
+11.7 (c 0.1, H2O) [Ref. (Wang et al.,
(KBr) nmax: 3462, 2976, 1732, 1453 and 1076 cmꢃ1; ESI–MS
(positive mode) m/z 1138.1 [M + Na]+. HR ESI–MS (positive mode)
m/z 1137.5468 [M + Na]+ (calcd for C55H86O23Na, 1137.5458); 1H
(400 MHz, C5D5N) and 13C NMR (75 MHz, C5D5N) data see Table 1.
2010) ½a 2D5
ꢂ
+48.0, H2O]. 1H NMR (
b-anomer, major, D2O) dH: 1.05
(3H, d, J = 6.4 Hz), 1.52 (1H, m), 1.87 (1H, m), 3.13 (1H, dd, J = 9.6,
3.0 Hz), 3.65 (1H, m), 3.92 (1H, d, J = 3.5 Hz), 4.91 (1H, dd, J = 10.3,
2.0 Hz). 13C NMR (
b-anomer, D2O) dC: 18.0, 39.0, 68.1, 70.1, 73.0,
2.3.6. Stauntoside T (6)
92.1. The data were consistent with those of
et al., 2010).
D-digitoxose (Hidekazu
White amorphous powder; ½a D28
ꢃ48.7 (c 0.5, CH3OH); IR (KBr)
ꢂ
nmax: 3507, 2938, 1540, 1455 and 1061 cmꢃ1; ESI-MS (positive
mode) m/z 961.6 [M + Na]+. HR ESI-MS (positive mode) m/z
961.5137 [M + Na]+ (calcd for C49H78O17Na, 961.5137); 1H (300 MHz,
C5D5N) and 13C NMR (75 MHz, C5D5N) data see Table 1.
D
-thevetose was obtained as a colorless gum with the following
analytical properties: ½a D27
ꢂ
+19.9 (c 0.3, H2O) [Ref. (Liu et al., 2007)
½
a 2D5 +3.5, H2O]. 1H NMR (
ꢂ
b-anomer, D2O, 600 MHz) dH: 1.25 (3H, d,
J = 6.3 Hz), 3.02 (1H, m), 3.03 (1H, m), 3.17 (1H, t, J = 8.6 Hz), 3.31
(1H, m), 3.62 (3H, s), 4.45 (1H, d, J = 7.9 Hz); 1H NMR (
-anomer,
a
2.4. Determination of absolute configuration of sugars
D2O, 600 MHz) dH: 1.20 (3H, d, J = 6.3 Hz), 2.99 (1H, m), 3.32 (1H,
overlap), 3.40 (1H, d, J = 3.8 Hz), 3.85 (1H, m), 3.63 (3H, s), 5.01 (1H,
d, J = 3.8 Hz). 13C NMR (
b
-anomer, D2O, 150 MHz) dC: 18.2, 61.3,
73.3, 76.4, 76.8, 87.7, 98.1; 13C NMR (
-anomer, D2O, 150 MHz) dC
18.3, 61.1, 68.4, 74.0, 77.3, 84.8, 94.0. The data were consistent with
those of -thevetose (Li et al., 2015).
-canarose was obtained as a colorless gum with the following
27 +58.5 (c 0.5, H2O) [Ref. (Toshiyuki et al.,
+45.7, H2O]. 1H NMR (
-anomer, CD3OD, 400 MHz) dH
2.4.1. Acid hydrolysis of compounds 2, 4, and 5 and determination of
the absolute configuration of sugars
a
:
Each solution of compounds 2, 4, and 5 (20 mg) in CH3OH (2 mL)
was refluxed with 10% hydrochloric acid (HCl) (3 mL) at 75 ꢄC for
2.5 h. After cooling, the reaction mixture was extracted thoroughly
with chloroform (CHCl3) (3 mL ꢀ 2), and the aqueous layer was
neutralized with barium hydroxide [Ba(OH)2] saturated with H2O.
The precipitates were filtered out, and the solution was dried to
yield a crude sugar fraction. The crude sugar fraction was subjected
to silica gel CC, successively eluting with CHCl3:CH3OH at the ratios
indicated to yield monosugars. For compound 2, cymarose (97:3,
v/v), digitoxose (93:7, v/v), canarose (9:1, v/v), and glucose (0:100,
v/v) were obtained from the crude sugar fraction; for compound 4,
diginose (98:2, v/v), cymarose (97:3, v/v), digitoxose (93:7, v/v),
thevetose (85:15, v/v), and glucose (0:100, v/v) were obtained from
the crude sugar fraction; and for compound 5, diginose (98:2, v/v),
cymarose (97:3, v/v), thevetose (85:15, v/v), and glucose (0:100,
v/v) were obtained from the crude sugar fraction.
D
D
analytical properties: ½a D
ꢂ
1997) ½a 2D0
ꢂ
b
:
1.27 (3H, d, J = 6.3 Hz), 1.47 (1H, m), 2.13 (1H, dd, J = 12.5, 5.3 Hz),
2.90 (1H, t, J = 9.0 Hz), 3.26 (1H, m), 3.50 (1H, m), 4.75 (1H, dd,
J = 9.6, 1.8 Hz); 1H NMR (
a-anomer, CD3OD, 400 MHz) dH: 1.22 (3H,
d, J = 6.3 Hz), 1.58 (1H, td, J = 12.2, 3.6 Hz), 2.03 (1H, dd, J = 12.8,
5.2 Hz), 2.92 (1H, t, J = 9.6 Hz), 3.81 (1H, m), 3.84 (1H, m), 5.19 (1H,
bd, J = 3.5 Hz). 13C NMR (
b
-anomer, CD3OD, 100 MHz) dC: 18.2, 42.0,
72.2, 73.3, 78.4, 94.8; 13C NMR (
-anomer, CD3OD, 100 MHz) dC
18.3, 39.8, 68.7, 69.4, 79.2, 92.6. The data were consistent with
those of -canarose (Toshiyuki et al., 1997).
a
:
D
L
-cymarose was obtained as a colorless gum with the following
analytical properties: ½a D27
ꢃ10.1 (c 0.1, H2O) [Ref. (Brasholz and
ꢂ
According to the procedure described previously (Tanaka et al.,
2007), the absolute configurations of glucoses were defined using
HPLC by comparing the retention times of derivatives of glucoses
obtained from hydrolyzing the compounds with those of stand-
ards. The monosugars obtained by acid hydrolysis as described
Reißig, 2009) ½a D22
ꢂ
ꢃ49.8, H2O]. 1H NMR (
b-pyranose, CD3OD,
400 MHz,) dH: 1.23 (3H, d, J = 6.7 Hz), 3.43 (3H, s), 4.95 (1H, dd,
J = 9.7, 2.5 Hz). 13C NMR (CD3OD, 100 MHz) dC: 18.8, 36.8, 58.1, 71.6,
74.6, 79.4, 93.1. 1H NMR (
b-furanose, CD3OD, 400 MHz) dH: 1.22
above were dissolved in pyridine and reacted with L-cysteine
(3H, d, J = 6.3 Hz), 3.30 (3H, s), 5.49 (1H, dd J = 5.4, 2.0 Hz); 13C NMR
(CD3OD, 100 MHz,) dC: 19.5, 41.2, 57.1, 69.3, 82.6, 89.6, 99.9. The
methyl ester hydrochloride at 60 ꢄC for 1 h. Then, arylisothiocya-
nates were added to the reaction mixtures, which were then stirred
at 60 ꢄC for another 1 h. The reaction mixtures were analyzed using
HPLC with a UV detector at 250 nm. Sugar standards were
derivatized in the same manner. The retention times (min) of
data were consistent with those of
Reißig, 2009).
L-cymarose (Brasholz and
D
-cymarose was obtained as a colorless gum with the following
analytical properties: ½a D27
ꢂ
+31.3 (c 0.1, H2O) [Ref. (Liu et al., 2007)
the derivatized standards were as follows: 19.26 min (
D-glucose)
½
a 2D5 + 47.6, H2O]. 13C NMR (
ꢂ
b-pyranose, CD3OD, 100 MHz) dC: 18.8,
and 17.94 min ( -glucose). By comparing their retention times with
L

42
C.-Z. Lai et al. / Phytochemistry Letters 16 (2016) 38–46
36.8, 58.1, 71.6, 74.6, 79.4, 93.1; 13C NMR (
b-furanose, CD3OD,
2.5. Cell culture
100 MHz) dC: 19.5, 41.2, 57.1, 69.3, 82.6, 89.6, 99.9. The data were
The RAW264.7 murine macrophage cell line was obtained from
American Type Culture Collection (ATCC, Manassas, VA). Cells were
cultured in complete DMEM medium supplemented with 10%
heat-inactivated FBS, penicillin G (100 units/mL), streptomycin
consistent with those of
Reißig, 2009).
D-cymarose (Liu et al., 2007; Brasholz and
L
-diginose was obtained as a colorless gum with the following
analytical properties: ½a D27
ꢃ24.8 (c 0.1, H2O) [Ref. (Brasholz and
ꢂ
(100 mg/mL), and L-glutamine (2 mM) (Gibco BRL Co, Grand Island,
Reißig, 2009) ½a D25
ꢂ
ꢃ66.0, H2O]. 13C NMR (
b-anomer, CD3OD,
NY) at 37 ꢄC in a humidified atmosphere containing 5% CO2 and 95%
100 MHz) dH: 16.2, 32.6, 54.9, 66.0, 70.7, 77.3, 93.8; 13C NMR
air.
(a-anomer, CD3OD, 100 MHz) dH: 16.3, 29.7, 55.2, 66.7, 67.1, 74.2,
91.5. The data were consistent with those of
and Reißig, 2009).
L-diginose (Brasholz
2.6. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2
(COX-2) expression bioassay
D
-oleandrose was obtained as
a
colorless gum with the
following analytical properties: ½a D27
ꢂ
+36.4 (c 0.1, H2O) [Ref.
Cells were cultured in six-well plates at a density of 4 ꢀ105
+20.8, H2O]. 13C NMR (
b-anomer, CD3OD,
cells/well and incubated for 24 h at 37 ꢄC. Next, cells were
(Shibano et al., 2012) ½a D
ꢂ
100 MHz) dH: 18.5, 38.8, 57.5, 73.5, 79.4, 81.9, 95.1. 13C NMR
-anomer, CD3OD, 100 MHz) dH: 18.5, 36.6, 57.5, 69.0, 77.9, 79.1,
pretreated with 33 mM of the compounds to be tested (1–14)
(
a
for 1 h and were stimulated with LPS (100 ng/mL) for another 18 h.
The cell culture medium was removed, and cells were washed
twice with cold PBS. Cells were subsequently incubated in RIPA
92.8. The data were consistent with those of
(Brasholz and Reißig, 2009).
D-oleandrose
Fig. 1. Structures of compounds 1–6.

C.-Z. Lai et al. / Phytochemistry Letters 16 (2016) 38–46
43
rotations of digitoxose (½a D27
ꢂ
+11.7) and oleandrose (½a D27
ꢂ
+36.4)
lysis buffer (Cell Signaling Technology, Boston, MA) containing
1 ꢀ protease inhibitor mix (Roche Applied Science) on ice for
30 min. The resulting cell solution was centrifuged at 10,000g for
20 min, and the supernatant was immediately collected and
transferred. The protein concentration of each cell lysate was
determined with a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA).
Forty micrograms of protein from each sample was separated via
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) followed by protein transfer to a nitrocellulose
membrane (GE Healthcare Life Sciences, Buckinghamshire). The
membrane was blocked in 5% skim milk for 1 h at room
temperature and then incubated with the corresponding primary
antibody. Antibodies for COX-2 and iNOS were obtained from Cell
were obtained from an aqueous solution after 24 h of equilibration.
The d-configurations of digitoxose and oleandrose were verified by
comparison of the experimental and reported specific rotations
(Shibano et al., 2012; Wang et al., 2010). The splitting patterns of
anomeric proton signals indicated that 1 has two sugar units with
b-linkages (Lin et al., 1995). The HMBC correlation from dH 5.58
(H-100) to dC 83.7 (C-40) suggested that digitoxopyranose was linked
to the C-40 position of oleandropyranose. The HMBC correlation
between dH 4.84 (1H, dd, J = 9.7, 1.8 Hz, H-10) and dC 78.0 (C-3)
revealed that the sugar chain was attached at C-3 of the aglycone,
which was confirmed by glycosylation shifts of the aglycone at C-2
(ꢃ2.0), C-3 (+ 6.6), and C-4 (ꢃ3.8) compared with glaucogenin C
(Nakagawa et al., 1983a,b,c). The 1H- and 13C NMR data of 1 were
assigned by the 1H- and 13C NMR, 1H-1H COSY, HSQC, and HMBC
spectra (Table 1). Thus, the structure of 1 was established as
Signaling Technology, Inc. (Boston, MA). Antibody for b-actin was
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
The antigen-antibody complexes were subsequently probed with
IRDye 800CW-conjugated goat anti-mouse IgG (H + L) or IRDye
800CW-conjugated goat anti-rabbit IgG (H + L) secondary anti-
bodies (Li-COR, Lincoln, NE).
glaucogenin C 3-O-
pyranoside, named “stauntoside O”.
b
-d-digitoxopyranosoyl-(1 !4)-
b-d-oleandro-
Stauntoside P (2) had the molecular formula of C46H70O19
,
determined by the pseudomolecular ion at m/z 949.4405 [M + Na]+
3. Results and discussion
in the positive HR ESI-MS experiment. The IR spectrum showed the
absorption bands for hydroxyl (3447 cmꢃ1
) and carbonyl
3.1. Chemistry
(1739 cmꢃ1) groups. The 1H- and 13C NMR data were assigned
by analyses of 1H-1H COSY, HSQC, HMBC and HSQC-TOCSY spectra
(Table 1). The aglycone of 2 was the same as that of 1 by
comparison of their 1H- and 13C NMR data. The 1H NMR spectrum
of 2 showed four anomeric proton signals at dH 4.90 (1H, d,
J = 9.9 Hz, H-10), 5.02 (1H, d, J = 7.3 Hz, H-10000), 5.04 (1H, br s, H-1000),
and 5.29 (1H, d, J = 9.5 Hz, H-100), suggesting that it contained four
sugar units in its structure. The presence of three secondary methyl
proton signals at dH 1.33 (3H, d, J = 6.1 Hz, H-600), 1.37 (3H, d,
J = 6.6 Hz, H-6000), and 1.39 (3H, d, J = 6.0 Hz, H-60) revealed that
three of them were 6-deoxysugars. By comparing its 1H- and 13C
Compounds 1–6 were obtained as white amorphous powders
and showed positive reactions to the Liebermann-Burchard and
Keller-Kiliani tests, suggesting that they were steroidal glycosides
with 2-deoxysugar units in their structures (Fig. 1).
Stauntoside O (1) was determined to possess the molecular
formula C34H50O11 by its pseudomolecular ion at m/z 657.3260
[M + Na]+ in the positive HR ESI-MS experiment. The IR spectrum
showed the absorption bands for hydroxyl (3443 cmꢃ1), carbonyl
) )
(1735 cmꢃ1 and olefinic (1516 cmꢃ1 groups. The 1H NMR
spectrum of 1 revealed the presence of two tertiary methyl
protons [dH 0.86 (3H, s, H-19) and 1.57 (3H, s, H-21)], two olefinic
protons [dH 5.43 (1H, d, J = 5.4 Hz, H-6) and 6.52 (1H, s, H-18)], two
oxygen-substituted methine protons [dH 3.79 (1H, m, H-3) and 5.48
NMR data with those of Komaroside G and glaucogenin C 3-O-
a-L-
cymaropyranosyl-(1 !4)- -digitoxopyranosyl- -canaropyr-
b
-
D
b-D
anoside (14) (Wang et al., 2004; Fu et al., 2007), the sugars in 2
were determined to be canarose, digitoxose, cymarose, and
glucose. The monosugars were obtained by acid hydrolysis of 2
and subsequent silica gel column chromatography purification.
The d-configuration of glucose was determined by acid hydrolysis
and appropriate derivatization of the resulting sugar (Tanaka et al.,
(1H, m, H-16)] and methylene protons [dH 3.98 (m, H-15
b) and 4.28
(m, H-15
a
)]. Comparison of the 1H- and 13C NMR data with those of
cynatratoside B (Sugama et al., 1986) revealed that 1 contained the
aglycone of glaucogenin C, which was confirmed by analyses of the
1H-1H COSY and HMBC spectra (Fig. 2). The 1H NMR spectrum
showed two anomeric protons [dH 4.84 (1H, dd, J = 9.7, 1.8 Hz, H-10)
and 5.58 (1H, dd, J = 9.7, 1.8 Hz, H-100)] and two secondary methyl
groups [dH 1.50 (3H, d, J = 5.6 Hz, H-60) and 1.61 (3H, d, J = 6.2 Hz,
H-600)], indicating that it contained two 6-deoxysugars in the
structure. The sugars were determined to be oleandrose and
digitoxose by comparing their 1H- and 13C NMR data with those of
corresponding deoxysugar units of glaucoside D and stauntoside K
(Nakagawa et al., 1983a,b,c; Yu et al., 2013a,b). The specific
2007). The absolute configurations of
D-canarose, D-digitoxose, and
L-cymarose were identified according to their specific rotation
values (Wang et al., 2010; Toshiyuki et al., 1997; Brasholz and
Reißig, 2009). The splitting patterns of anomeric proton signals
indicated that the l-cymarose was in an
three sugars were in
a-linkage, and the other
b
-linkages (Lin et al., 1995). The 1 !4 linkage
of the sugars was determined by the HMBC correlations from dH
5.02 (H-10000) to dC 78.5 (C-4000), from dH 3.43 (H-400) to dC 98.9
(C-1000), and from dH 5.29 (H-100) to dC 89.0 (C-40). The HMBC
Fig. 2. Key 1H-1H COSY and HMBC correlations of 1.

44
C.-Z. Lai et al. / Phytochemistry Letters 16 (2016) 38–46
correlation from dH 4.90 (H-10) to dC 78.1 (C-3) revealed that the
sugar chain was linked to C-3 of the aglycone. Thus, the structure of
2007; Brasholz and Reißig, 2009). The absolute configuration of
glucose was determined to be d by acid hydrolysis and appropriate
derivatization of the resulting sugar (Tanaka et al., 2007). The
1 !4 linkage of sugar units was determined by the HMBC
correlations from dH 4.99 (H-100000) to dC 74.8 (C-40000), from dH
5.20 (H-10000) to dC 82.8 (C-4000), from dH 5.14 (H-1000) to dC 83.6
(C-400), and from dH 5.53 (H-100) to dC 83.4 (C-40). The HMBC
correlation from dH 4.84 (H-10) to dC 78.7 (C-3) revealed that
the sugar chain was attached at C-3 of the aglycone. The
splitting patterns of anomeric proton signals indicated that only
2 was determined to be glaucogenin C 3-O-
b
-
D
-glucopyranosoyl-
-digitoxopyranosoyl-
D-canaropyranoside, named “stauntoside P”.
(1 !4)-
a
-
L
-cymaropyranosoyl-(1 !4)-
b-D
(1 !4)-
b-
Stauntoside Q (3) was determined to possess the molecular
formula C49H76O18 by its pseudomolecular ion at m/z 975.4924
[M + Na]+ in the positive HR ESI-MS experiment. The 1H- and 13C
NMR data were assigned by analyses of 1H-1H COSY, HSQC, HMBC,
and HSQC-TOCSY spectra (Table 1). Comparison of its 1H- and 13
C
NMR data with those of 1 revealed that they had the same aglycone
and that the difference lay in the sugar chain. Comparison of its 1H-
and 13C NMR data with those of stauntoside G (Yu et al., 2013a,b)
suggested that they had the same sugar chain, which was
confirmed by analyses of 1H-1H COSY, HSQC, HMBC and HSQC-
TOCSY experiments. The L configuration of diginose and D
configurations of cymarose and thevetose were determined
according to their specific rotation values after mild acid hydrolysis
of 3 and subsequent silica gel CC purification (Liu et al., 2007;
Brasholz and Reißig, 2009). The sequence of the sugars was
l-diginopyranose was in an
sugars were in -linkages in 4 (Lin et al., 1995). Hence, 4 was
identified as glaucogenin C 3-O-
-glucopyranosoyl-(1 !4)-
-cymaropyranosoyl-(1 !4)-
-thevetopyranoside,
a-linkage and that the other four
b
b
-D
a-L-
diginopyranosoyl-(1 !4)-
b
-
b
D
b-D-
digitoxopyranosoyl-(1 !4)-
-D
named
“stauntoside R”.
The molecular formula of stauntoside S (5) was determined to
be C55H86O23 by its pseudomolecular ion at m/z 1137.5468
[M + Na]+. The 1H- and 13C NMR data were assigned by analyses
of 1H-1H COSY, HSQC, HMBC and HSQC-TOCSY spectra (Table 1).
Comparison of its 1H- and 13C NMR data with those of 1 revealed
that they had the same aglycone. The presence of five anomeric
proton signals at dH 4.83 (1H, d, J = 7.7 Hz, H-10), 4.97 (1H, d,
J = 7.7 Hz, H-100000), 5.08 (1H, d, J = 9.6 Hz, H-1000), 5.22 (1H, d,
J = 2.4 Hz, H-10000), and 5.31 (1H, d, J = 9.5 Hz, H-100) in the 1H NMR
spectrum indicated that it contained five sugar units. The presence
of four secondary methyl proton signals at dH 1.37 (3H, d, J = 6.2 Hz,
H-600), 1.39 (3H, d, J = 6.4 Hz, H-6000), 1.48 (3H, d, J = 3.0 Hz, H-60), and
1.71 (3H, d, J = 6.3 Hz, H-60000) suggested that four of them were 6-
deoxysugars. The 1H- and 13C NMR data of 5 were almost identical
with those of 4 except that the digitoxose was replaced by
cymarose. The deoxysugars of d-cymarose, d-thevetose, and l-
diginose were determined by their specific rotation values after
mild acidic hydrolysis of 5 and subsequent silica gel CC purification
(Liu et al., 2007; Brasholz and Reißig, 2009). The d-glucose in 5 was
determined by acid hydrolysis and appropriate derivatization of
the resulting sugar (Tanaka et al., 2007). The splitting patterns of
anomeric proton signals indicated that only l-diginopyranose was
confirmed by the HMBC correlations from dH 5.24 (H-10000 of
L-
diginopyranose) to dC 82.6 (C-4000), from dH 5.10 (H-1000 of d-
cymaropyranose) to dC 83.7 (C-400), and from dH 5.33 (H-100 of d-
cymaropyranose) to dC 83.1 (C-40). The HMBC correlation from dH
4.83 (H-10 of d-thevetopyranose) to dC 78.7 (C-3) revealed that the
sugar chain was attached at the C-3 of the aglycone. The splitting
patterns of anomeric proton signals indicated that the l-
diginopyranose was in an
sugars were in -linkages (Lin et al.,1995). Thus, 3 was determined
to be glaucogenin C 3-O- -cym-
-diginopyranosoyl-(1 !4)-
aropyranosoyl-(1 !4)- -cymaropyranosoyl-(1 !4)- -theve-
topyranoside, named “stauntoside Q”.
The positive HR ESI-MS of stauntoside R (4) showed
a-linkage and that the other three
b
a
-L
b-D
b-D
b-D
a
pseudomolecular ion at m/z 1123.5300 [M + Na]+, corresponding
to the molecular formula C54H84O23. The 1H- and 13C NMR data
were assigned by analyses of 1H-1H COSY, HSQC, HMBC and HSQC-
TOCSY spectra (Table 1). Comparison of its 1H- and 13C NMR data
with those of 1 revealed that they had the same aglycone and
differed in the sugar chain. The 1H NMR spectrum of 4 showed five
anomeric proton signals at dH 4.84 (1H, d, J = 7.8 Hz, H-10), 4.99 (1H,
d, J = 7.8 Hz, H-100000), 5.14 (1H, dd, J = 9.6, 1.2 Hz, H-1000), 5.20 (1H, d,
J = 3.2 Hz, H-10000), and 5.53(1H, dd, J = 9.6, 1.2 Hz, H-100), suggesting
that it contained five sugar units in the structure. Four secondary
methyl proton signals at dH 1.31 (3H, d, J = 6.0 Hz, H-6000),1.43 (3H, d,
J = 6.0 Hz, H-600), 1.48 (3H, d, J = 6.0 Hz, H-60), and 1.71 (3H, d,
J = 6.4 Hz, H-60000) revealed the presence of four 6-deoxysugars. The
sugar units were identified as thevetose, digitoxose, cymarose,
diginose, and glucose by comparing their 1H and 13C NMR data
with those found in the literature (Yu et al., 2013a,b; Nakagawa
et al., 1983a,b,c). The l configuration of diginose and d config-
urations of digitoxose, cymarose and thevetose were determined
according to their specific rotations after acid hydrolysis of 4 and
subsequent silica gel CC purification (Wang et al., 2010; Liu et al.,
in an
-linkages (Lin et al.,1995). The HMBC correlation from dH 4.83 (H-
10) to dC 78.5 (C-3) revealed that the sugar chain was attached to C-
3 of aglycone. Thus, 5 was identified to be glaucogenin C 3-O-
glucopyranosoyl-(1 !4)- -diginopyranosoyl-(1 !4)- -cym-
aropyranosoyl-(1 !4)- -cymaropyranosoyl-(1 !4)- -theve-
topyranoside, named “stauntoside S”.
a-linkage and that the other four sugars in 5 were in
b
b-D-
a
-L
b-D
b
-D
b-D
The molecular formula of stauntoside T (6) was determined to
be C49H78O17 by its pseudomolecular ion at m/z 961.5137 [M + Na]+.
The IR spectrum showed the absorption bands for hydroxyl
(3507 cmꢃ1) and olefinic (1540 cmꢃ1) groups. The 1H-and 13C NMR
data were assigned by analyses of 1H-1H COSY, HSQC, HMBC and
HSQC-TOCSY experiments (Table 1). The 1H NMR spectrum of 6
showed the presence of three tertiary methyl protons at dH 0.93
(3H, s, H-19), 1.10 (3H, s, H-18), and 1.74 (3H, s, H-21). Comparison
Fig. 3. Key 1H-1H COSY and HMBC correlations of the aglycone of 6.

C.-Z. Lai et al. / Phytochemistry Letters 16 (2016) 38–46
45
Fig. 4. Inhibitory effects of compounds 1–14 on LPS-induced iNOS and COX-2 expression in RAW264.7 macrophages.
of its 1H and 13C NMR data with those of argeloside C revealed
3.2. Bioactivity
that they had the same aglycone and that the difference lay in the
sugar chain (Plaza et al., 2005), which was confirmed by analyses
of the 1H-1H COSY and HMBC spectra (Fig. 3). In the ROESY
spectrum, H-8 (dH 1.96, 1H, m) correlated with H-18 (dH 1.10, 3H, s)
Immune cells, including macrophages and monocytes, are
involved in various inflammatory diseases, which are pathophy-
siologically complex (Mcinnes and Schett, 2011; Galkina and Ley,
2009; Vergani and Mieli, 2008). Lipopolysaccharide (LPS) can
activate macrophages to produce proinflammatory mediators,
including nitric oxide (NO) and prostaglandin E2 (PGE2) (Chao
et al., 2008). Both NO and PGE2 are among the most prominent
inflammatory mediators. NO is induced by NO synthase (iNOS),
and prostaglandins are regulated by cyclooxygenase-2 (COX-2;
prostaglandin H2 synthase). Given the key role of macrophages
in inflammation, in our current study, the anti-inflammatory
effects of compounds 1–14 were investigated by measuring their
ability to inhibit protein expression of iNOS and COX-2 in
RAW246.7 cells stimulated by LPS (Fig. 4). Dexamethasone
(DEX) was used as a positive control at a working concentration
and H-19 (dH 0.93, 3H, s), while H-1a (dH 1.11, 1H, m) correlated
with H-3 (dH 3.84, 1H, m) and H-9 (dH 1.65, 1H, m); this suggested a
trans BC ring junction, while 18- and 19-CH3 groups had axial
orientation. The small J value of H-17 (2.6 Hz) revealed the cis DE
ring junction. Thus, the relative configuration of the aglycone for 6
was determined to be the same as previously reported (Plaza et al.,
2005; Perrone et al., 2006). Based on the generally accepted
b
-orientation of CH3-19 in 14,15-secopregnane-type skeletons,
the aglycone was determined to be (14S,16S,20R)-14,16–14,20–
15,20-triepoxy-14,15-secopregn-5-en-3-ol. Comparison of the
1H- and 13C NMR data with those of 3 suggested that they had
the same sugar chain in the structure, which was confirmed by
analyses of 1H-1H COSY, HSQC, HMBC and HSQC-TOCSY spectra.
of 0.5 mM. Pharmacological results revealed that compounds 1, 5,
The absolute configurations of d-cymarose, d-thevetose, and
L
-
8, 9, 11, and 13 could clearly inhibit protein expression of iNOS,
while compounds 5 and 7 could, to a certain extent, decrease COX-
2 protein expression in LPS-stimulated RAW246.7 cells compared
to LPS-stimulated cells that received no other treatment. Based on
these results, we speculate that these compounds affected the
release of NO and/or PGE2, respectively. Compound 14 could
dramatically inhibit iNOS and COX-2 protein expression compared
to LPS stimulation alone; however, it also showed strong
diginose were determined by their specific rotations after mild
acidic hydrolysis of 6 and subsequent silica gel CC purification
(Liu et al., 2007; Brasholz and Reißig, 2009). The 1 !4 linkage
of the sugars was confirmed by the HMBC correlations from
dH 5.24 (H-10000 of l-diginopyranose) to dC 82.7 (C-4000), from dH 5.10
(H-1000 of d-cymaropyranose) to dC 83.7 (C-400), from dH 5.32 (H-100
of d-cymaropyranose) to dC 83.3 (C-40), and from dH 4.83 (H-10 of
D-
thevetopyranose) to dC 78.7 (C-3). The splitting patterns of
cytotoxicity at a concentration of 33 mM. Therefore, we considered
anomeric proton signals indicated that the l-diginopyranose
this inhibitory effect on iNOS and COX-2 to be related to the
cytotoxic effects. Our results indicate that compounds 1, 5, 7–9, 11,
and 13 have the potential to medicate anti-inflammatory effect,
with compound 5 having a stronger anti-inflammatory effect than
other compounds.
was
et al., 1995). Thus, 6 was determined to be (14S,16S,20R)-14,16–
14,20–15,20-triepoxy-14,15-secopregn-5-en-3-ol-3-O- -digino-
pyranosyl-(1 !4)- -cymaropyranosyl-(1 !4)- -cymaropyr-
anosyl-(1 !4)- -thevetopyranoside, named “stauntoside T”.
a-linkage, and the other three sugars were b-linkages (Lin
a-L
b-
D
b-D
b-D
The eight known pregnane glycosides were cynatratoside D (7)
(Wang et al., 2007), stauntoside L (8) (Yu et al., 2013a,b),
stauntoside M (9) (Yu et al., 2013a,b), cynatratoside B (10) (Sugama
et al., 1986), glaucogenin C mono-d-thevetoside (11) (Nakagawa
et al., 1983a,b,c), glaucoside G (12) (Nakagawa et al., 1983a,b,c),
Acknowledgments
We are grateful to the State Key Laboratory of Quality
Research in Chinese Medicine, Macau University of Science and
Technology for the anti-inflammatory activity testing. Associate
Professor Ying Zhang’s help in plant authentication is also
appreciated. We are also grateful to Mr. T. Shi, Mr. J. Geng, and Dr.
Y. Yu for the HR ESI-MS and NMR measurements. The major
chemical work was accomplished in Guangzhou Xiangxue
Pharmaceutical Ltd.
stauntoside B (13) (Yu et al., 2013a,b) and glaucogenin C 3-O-
a-L-
cymaropyranosyl-(1 !4)-
b
-
D
-digitoxopyranosyl- -canaropyra-
b-D
noside (14) (Fu et al., 2007). The identification of these substances
was established by comparing their physical and spectroscopic
data with those reported previously.

46
C.-Z. Lai et al. / Phytochemistry Letters 16 (2016) 38–46
thevetoside from the Chinese drug Pai-ch’ien Cynanchum glaucescens Hand-
References
Mazz. Chem. Pharm. Bull. 31, 870–878.
Nakagawa, T., Hayashi, K., Mitsuhashi, H., 1983c. Studies on the constituents of
Asclepiadaceae plants. LIV. The structures of glaucoside-F and -G from the
Chinese drug Pai-ch’ien: Cynanchum glaucescens Hand-Mazz. Chem. Pharm.
Bull. 31, 879–882.
Perrone, A., Plaza, A., Ercolino, S.F., Hamed, A.I., Parente, L., Pizza, C., Piacente, S.,
2006. 14,15-Secopregnane derivatives from the leaves of Solenostemma argel. J.
Nat. Prod. 69, 50–54.
Plaza, A., Perrone, A., Balestrieri, C., Balestrieri, M.L., Bifulco, G., Carbone, V., Hamed,
A., Pizza, C., Piacente, S., 2005. New antiproliferative 14,15-secopregnane
glycosides from Solenostemma argel. Tetrahedron 61, 7470–7480.
Shibano, M., Misaka, A., Sugiyama, K., Taniguchi, M., Baba, K., 2012. Two
secopregnane-type steroidal glycosides from Cynanchum stauntonii (Decne.)
Schltr.ex Levl. Phytochem. Lett. 5, 304–308.
Sugama, K., Hayashi, K., Mitsuhashi, H., Kaneko, K.,1986. Studies on the constituents
of Asclepiadaceae plants. LXVI. The structures of three new glycosides,
cynapanosides A, B, and C, from the Chinese drug Xu-Chang-Qing, Cynanchum
paniculatum Kitagawa. Chem. Pharm. Bull. 34, 4500–4507.
Tanaka, T., Nakashima, T., Ueda, T., Tomii, K., Kouno, I., 2007. Facile discrimination of
aldose enantiomers by reversed-phase HPLC. Chem. Pharm. Bull. 55, 899–901.
Toshiyuki, N., Tsutomu, F., Naoko, O., Wataru, H., Junko, N., Tadatake, O., 1997.
Synthesis of highly deoxidized monosaccharides from methyl 3-O-acetyl-6-O-
(p-tolylsulfonyl)-b-D-glucopyranoside. J. Appl. Glycosci. 44, 175–181.
Vergani, D., Mieli, V.G., 2008. Aetiopathogenesis of autoimmune hepatitis. World J.
Gastroenterol. 14, 3306–3312.
Wang, L.Q., Shen, Y.M., Xing, X., Wei, Y.Q., Zhou, J., 2004. Five new C21 steroidal
glycosides from Cynanchum komarovii Al. lljinski. Steroids 69, 319–324.
Wang, L.Q., Lu, Y., Zhao, Y.X., Zhou, J., Zheng, Q.T., 2007. Three new C21-steroidal
glycosides from the roots of Cynanchum inamoenum. J. Asian Nat. Prod. Res. 9,
771–779.
Wang, L., Yin, Z.Q., Wang, Y., Zhang, X.Q., Li, Y.L., Ye, W.C., 2010. Perisesaccharides A–
E: new oligosaccharides from the root barks of Periploca sepium. Planta Med. 76,
909–915.
Yu, J.Q., Deng, A.J., Qin, H.L., 2013a. Nine new steroidal glycosides from the roots of
Cynanchum stauntonii. Steroids 78, 79–90.
Yu, J.Q., Zhang, Z.H., Deng, A.J., Qin, H.L., 2013b. Three new steroidal glycosides from
the roots of Cynanchum stauntonii. Bio. Med. Res. Int. 816145.
Zhu, N.Q., Wang, M.F., Kikuzaki, H., Nakatani, N., Ho, C.T., 1999. Two C21-steroidal
glycosides isolated from Cynanchum stauntoi. Phytochemistry 52, 1351–1355.
Brasholz, M., Reißig, H.U., 2009. Alkoxyallene-based de novo synthesis of rare deoxy
sugars: new routes to
L-cymarose, L-sarmentose, L-diginose and L-oleandrose.
Eur. J. Org. Chem. 21, 3595–3604.
Chao, C.H., Wen, Z.H., Su, J.H., Chen, I., Huang, H.C., Dai, C.F., Sheu, J.H., 2008. Further
study on anti-inflammatory oxygenated steroids from the octocoral
Dendronephthya griffini. Steroids 73, 1353–1358.
Commission, C.P., 2010. Pharmacopoeia of the People’s Republic of China. China
Medical Science Press, Beijing, pp. 101.
Day, S.H., Wang, J.P., Won, S.J., Lin, C.N., 2001. Bioactive constituents of the roots of
Cynanchum atratum. J. Nat. Prod. 64, 608–611.
Fu, M.H., Wang, Z.J., Yang, H.J., Maimai, M., Fang, J., Tang, L.Y., Yang, L., 2007. A new
C21-steroidal glycoside from Cynanchum stauntonii. Chin. Chem. Lett. 18, 415–
417.
Galkina, E., Ley, K., 2009. Immune and inflammatory mechanisms of atherosclerosis.
Annu. Rev. Immunol. 27, 165.
Hidekazu, H., Hideaki, A., Keisuke, K., Hiroyuki, A., 2010. Nucleophilic addition to
2,3-disubstituted butanal derivatives and their application to natural product
synthesis. Chem. Pharm. Bull. 58, 1411–1418.
Li, Y.M., Wang, L.H., Li, S.L., Chen, X.Y., Shen, Y.M., Zhang, Z.K., He, H.P., Xu, W.B., Shu,
Y.L., Liang, G.D., Fang, R.X., Hao, X.J., 2007. Seco-pregnane steroids target the
subgenomic RNA of alphavirus-like RNA viruses. Proc. Natl. Acad. Sci. U. S. A 104,
8083–8088.
Li, J.L., Zhou, J., Chen, Z.H., Guo, S.Y., Li, C.Q., Zhao, W.M., 2015. Bioactive C21-
steroidal glycosides from the roots of Cynanchum otophyllum that suppress the
seizure-like locomotor activity of zebrafish caused by pentylenetetrazole. J. Nat.
Prod. 78, 1548–1555.
Lin, Y.L., Lin, T.C., Kuo, Y.H., 1995. Five new pregnane glycosides from Cynanchum
taiwanianum. J. Nat. Prod. 58, 1167–1173.
Liu, Y.B., Tang, W.Z., Yu, S.S., Qu, J., Liu, J., Liu, Y., 2007. Eight new C-21 steroidal
glycosides from Dregea sinensis var corrugata. Steroids 72, 514–523.
Mcinnes, I.B., Schett, G., 2011. The pathogenesis of rheumatoid arthritis. N. Engl. J.
Med. 365, 2205–2219.
Nakagawa, T., Hayashi, K., Wada, K., Mitsuhashi, H., 1983a. Studies on the
constituents of Asclepiadaceae plants—LII. The structures of five glycosides
glaucoside A, B, C, D, and E from Chinese drug Pai-ch’ien Cyanchum glaucescens
Hand-Mazz. Tetrahedron 39, 607–612.
Nakagawa, T., Hayashi, K., Mitsuhashi, H., 1983b. Studies on the constituents of
Asclepiadaceae plants. LIII. The structures of glaucogenin-A, -B, and -C mono-D-