Steroidal saponins from the roots and rhizomes of tupistra chinensis

Article abstract of DOI:10.3390/molecules200813659

Two new furostanol saponins 1-2 and a new spirostanol saponin 3 were isolated together with two known furostanol saponins 4-5 from the roots and rhizomes of Tupistra chinensis. Their structures were characterized as 1β,2β,3β,4β,5β,26-hexahydroxyfurost-20(22), 25(27)-dien-5,26-O-β-D-glucopyranoside (1), 1β,2β,3β,4β,5β,6β,7α,23ξ,26-nona-hydroxyfurost- 20(22),25(27)-dien-26-O-β-D-glucopyranoside (2), (20S,22R)-spirost-25 (27)-en-1β,3β,5β- trihydroxy-1-O-β-D-xyloside (3), tupisteroide B (4) and 5β-furost-Δ25(27)-en-1β,2β,3β,4β,5β,7α, 22ξ,26-octahydroxy-6-one-26-O-β-D-glucopyranoside (5), respectively, by extensive use of spectroscopic techniques and chemical evidence. Additionally, the in vitro cytotoxic activity of 1-4 was evaluated on human A549 and H1299 tumor cell lines, and compound 3 exhibited cytotoxicity against A549 cells (IC50 86.63 ± 2.33 μmol·L-1) and H1299 cells (IC50 88.21 ± 1.34 μmol·L-1).

Full text of DOI:10.3390/molecules200813659

Molecules 2015, 20, 13659-13669; doi:10.3390/molecules200813659
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Steroidal Saponins from the Roots and Rhizomes of
Tupistra chinensis
1
,†
2,†
3
1
1
1
1
Yuze Li , Xin Wang , Hao He , Dongdong Zhang , Yi Jiang , Xinjie Yang , Fei Wang ,
1
1,
1,
Zhishu Tang , Xiaomei Song * and Zhenggang Yue *
1
Shaanxi Collaborative Innovation Center of Chinese Medicinal Resource Industrialization,
Shaanxi Province Key Laboratory of New Drugs and Chinese Medicine Foundation Research,
Shaanxi Rheumatism and Tumor Center of TCM Engineering Technology Research,
School of Pharmacy, Shaanxi University of Chinese Medicine, Xianyang 712046, China;
E-Mails: lyz1990yeah@163.com (Y.L.); zhangnatprod@163.com (D.Z.); j9668216@126.com (Y.J.);
xxx211xxx@126.com (X.Y.); wf88-88@163.com (F.W.); tzs6565@163.com (Z.T.)
2
The First Hospital of Xi’an, Xi’an 710002, China; E-Mail: wangxin.x@163.com
3
School of Pharmaceutical Sciences, Xi’an Medical University, Xi’an 710021, China;
E-Mail: hehao313@163.com
†
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed; E-Mails: songxiaom@126.com (X.S.);
liuxingjian1981@163.com (Z.Y.); Tel.: +86-136-3673-3632 (X.S.); +86-180-9208-6211 (Z.Y.).
Academic Editor: Derek J. McPhee
Received: 25 June 2015 / Accepted: 23 July 2015 / Published: 28 July 2015
Abstract: Two new furostanol saponins 1–2 and a new spirostanol saponin 3 were isolated
together with two known furostanol saponins 4–5 from the roots and rhizomes of Tupistra
chinensis. Their structures were characterized as 1β,2β,3β,4β,5β,26-hexahydroxyfurost-20(22),
25(27)-dien-5,26-O-β-D-glucopyranoside (1), 1β,2β,3β,4β,5β,6β,7α,23ξ,26-nona-hydroxyfurost-
2
0(22),25(27)-dien-26-O-β-D-glucopyranoside (2), (20S,22R)-spirost-25 (27)-en-1β,3β,5β-
trihydroxy-1-O-β-D-xyloside (3), tupisteroide B (4) and 5β-furost-Δ25(27)-en-1β,2β,3β,4β,5β,7α,
2ξ,26-octahydroxy-6-one-26-O-β-D-glucopyranoside (5), respectively, by extensive use of
2
spectroscopic techniques and chemical evidence. Additionally, the in vitro cytotoxic activity
of 1–4 was evaluated on human A549 and H1299 tumor cell lines, and compound 3 exhibited
−1
cytotoxicity against A549 cells (IC50 86.63 ± 2.33 μmol·L ) and H1299 cells (IC50 88.21 ±
−
1
1
.34 μmol·L ).

Molecules 2015, 20
Keywords: Tupistra chinensis; steroidal saponins; structure identification; cytotoxic activity
13660
1
. Introduction
Tupistra chinensis Baker., a species in the Tupistra genus of the Liliaceae family, is used as an endemic
herbal medicine, known as “Kai-Kou-Jian”, in the Qinba Mountains of Shaanxi Province in China [1]. The
roots and rhizomes of T. chinensis are commonly used as folk medicine to treat throat irritation, rheumatic
diseases and snake-bites [2,3]. Modern pharmacological experiments have showed that the extracts of
this species possess significant antitumor activities [4,5], moreover, two main kinds of components—
cardenolides and saponins—were isolated from T. chinensis [3,6,7]. As part of our research project to
find more diverse bioactive leading compounds from the medicinal herbs of the Qinba Mountains [8–11],
the chemical constituents and pharmacological studies of T. chinensis were investigated, and two new
furostanol saponins, 1β,2β,3β,4β,5β,26-hexahydroxyfurost-20(22),25(27)-dien-5,26-O-β-D-glucopyranoside
(1), 1β,2β,3β,4β,5β,6β,7α,23ξ,26-nonahydroxyfurost-20(22),25(27)-dien-26-O-β-D-glucopyranoside (2),
and a new spirostanol saponin (20S,22R)-spirost-25(27)-en-1β,3β,5β-trihydroxy-1-O-β-D-xyloside (3)
were obtained from the roots and rhizomes of T. chinensis together with the two known compounds
tupisteroide B (4) and 5β-furost-Δ25(27)-en-1β,2β,3β,4β,5β,7α,22ξ,26-octahydroxy-6-one-26-O-β-D-
glucopyranoside (5) (Figure 1). The cytotoxic activity of 1–4 was evaluated on human A549 and H1299
tumor cells.
Figure 1. Structures of compounds 1–5.
2
. Results and Discussion
Compound 1 was obtained as a white amorphous powder, which showed positive reactions in the
Liebermann-Burchard, Ehrlich and Molisch reactions, suggesting that 1 was a furostanol glycoside. Its
molecular formula was determined as C39
17 from the HR-ESI-MS peak at m/z 801.3855 [M − H]−.
62
H O

Molecules 2015, 20
13661
1
The H-NMR spectrum showed three methyl protons at δ
H
0.67 (3H, s), 1.70 (3H, s) and 1.58 (3H, s),
two exo-methylene protons (δ
protons at (δ 5.28 (d, J = 7.8 Hz) and 4.89 (1H, d, J = 7.7 Hz)). The C-NMR spectrum displayed 39
carbon signals, 27 of which belonged to the aglycone carbons, while the remaining signals were assignable
to two glucosyl moieties (δ 103.8, 75.8, 78.5, 71.7, 78.6 and 62.6, and δ 97.4, 76.2, 78.6, 71.9, 78.8
and 62.8). Among carbon signals of the aglycone, δ 146.2 and 111.6 were due to an olefinic bond group,
14.3, 13.7 and 11.7 were due to three methyl groups, and δ 77.8, 68.1, 75.2, 67.6, 87.4, 84.4, 64.5
H
5.35 (1H, brs) and 5.04 (1H, brs)), as well as signals for two anomeric
1
3
H
C
C
C
δ
C
C
and 71.7 were due to eight oxygenated carbon groups, which indicated that 1 was a furostanol saponin with
multiple hydroxyl groups. The structure of 1 was finally determined by analysis of its 2D NMR data (see
Figure 2). The HMQC experiment allowed for the assignments of the proton and protonated carbon
resonances in the NMR spectra of 1. HMQC correlations of (δ
H
5.35 (H-27a) and 5.04 (H-27b)) to δ 111.6,
C
showed the appearance of a terminal olefinic bond at C-27. Then, HMBC correlations of H-27/C-24, C-25
and C-26, H-24/C-22, C-23, C-25 and C-26, H-26/C-24, C-25 and C-27, indicated that the appearance
of an isopentene group, linked at C-22 of the tetrahydrofuran ring of the furostanol saponin. Moreover,
HMBC correlations of H-19/C-1, C-5, C-9 and C-10, H-3/C-1, C-2 and C-5, and H-6/C-4 and C-5,
indicated that all hydroxyl groups were linked at C-1–C-5 of the A ring of the furostanol saponin (see
Figure 2). Furthermore, the remaining HMBC correlations of H-18/C-12, C-13, C-14 and C-17, H-16/C-13,
C-17, C-20 and C-22, H-21/C-17, C-20 and C-22, were assigned (see Figure 2). Therefore, the aglycone
of 1 was identified as 1, 2, 3, 4, 5, 26-hexanol-furost-20 (22),25(27)-dien. In addition, the HMBC correlation
signals of H-Glc-1′/C-5 and H-Glc-1′′/C-26, indicated that glucosyl groups were connected as (Glc-1′′-
O-C-26) and (Glc-1′-O-C-5) (see Figure 2). The two glucosyl moieties were identified as D-glucose by
acid hydrolysis of 1, followed by TLC comparison with a reference compound and optical rotation
determination [12], and judged to be in a β-configuration [13] from the coupling constants of the anomeric
protons (7.8 Hz and 7.7 Hz, respectively). In the NOESY spectrum of 1, the NOE correlations of
Me-19/H-8, H-9/H-4, H-4/H-3 and H-2, and H-2/H-1 were observed (see Figure 2), indicated α-axial
configurations of H-1, H-2, H-3 and H-4, and β-orientation of Me-19, 1-OH, 2-OH, 3-OH, 4-OH and 5-OH,
which supported the A/B cis ring junction pattern; the NOE correlations of Me-19/H-8, H-8/Me-18,
and H-14/H-9, H-16 and H-17, supported the B/C and C/D trans ring junction pattern; and the NOE
correlations of Me-18/H-15b, H-15a/H-16 and H-17, and H-17/Me-21, suggested an α-orientation of
Me-21 (see Figure 2). Therefore, compound 1 was identified as 1β,2β,3β,4β,5β,26-hexahydroxyfurost-
2
0(22),25(27)-dien-5,26-O-β-D-glucopyranoside.
Figure 2. Key HMBC and NOESY correlations of the compound 1.

Molecules 2015, 20
13662
Compound 2 was obtained as a white amorphous powder, which showed positive reactions in
the Liebermann-Burchard, Ehrlich, and Molisch tests, suggesting that 2 was a furostanol glycoside. The
molecular formula C33
H
52
O
15 was deduced from the HR-ESI-MS peak at m/z 711.3198 [M + Na]+.
Comparison of the HR-ESI-MS and NMR data of 2 and 1, indicated almost similar NMR spectroscopic
1
3
features, except for the number of oxygenated methine groups. In the C-NMR spectrum of 2, only one
glucosyl moiety (δ 104.2, 75.6, 80.0, 72.1, 79.0, 63.2) was recognized, however, nine oxygenated
carbon groups of the aglycone at δ 79.1, 67.7, 76.1, 70.2, 78.6, 74.0, 72.5, 64.8 and 72.7 were identified.
C
C
Meanwhile, the spectroscopic features of 2 were similar to those of tupisteroide B (4), indicating that
seven hydroxyl groups were linked at C-1–C-7 of the furostanol saponin, which was confirmed by the
1
1
H- H COSY correlation of H-1/H-2/H-3/H-4 and H-6/H-7 and the HMBC correlation of H-19/C-1,
C-5, C-9 and C-10, and H-6/C-4 and C-5 (see Figure 3). The 26-OH was connected with the glucosyl
moiety from the correlation signals of H-Glc-1′/C-26 in the HMBC spectra (see Figure 3). The remaining
hydroxyl group was deduced to be linked at C-23, from one oxygen-bearing methine signal occurring at
δ
C
64.8 in 2, instead of a methylene carbon (C-23) at δ
C
34.3 in 4, which was correlated with a proton
signal at δ 5.13 (dd, J = 6.0, 8.0 Hz, H-23) in the HMQC spectrum, and the correlation signals of
H
1
1
H-23/H-24 in the H- H COSY spectrum, the correlation signals of H-23/C-20, C-22, C-24 and C-25,
H-24/C-22, C-23, C-25, C-26 and C-27, and H-27/C-24, C-25 and C-26 in the HMBC spectrum (see
Figure 3). In addition, the glucosyl moiety was identified as β-D-glucose by the acid hydrolysis procedure
and the coupling constant analysis of the anomeric proton (J = 7.8 Hz), according to the same protocol
as that described for 1. Thus, the planar structure of 2 was deduced as 1,2,3,4,5,6,7,23,26-nonanolfurost-
20(22),25(27)-dien-26-O-β-D-glucose. In the NOESY spectrum of 2, the NOE correlations of Me-19/H-8,
H-4/H-2, H-3 and H-9, and H-2/H-1 were observed, indicating α-axial configurations of H-1, H-2, H-3,
and H-4, and β-orientation of Me-19, 1-OH, 2-OH, 3-OH, 4-OH and 5-OH, which supported the A/B cis
ring junction pattern (see Figure 3). Besides, NOE correlation of H-7/H-8 was observed and no correlation
signals was occurred between Me-19/H-6, which indicated α-axial configuration of 7-OH and β-orientation
of 6-OH (see Figure 3). Finally, the NOE correlations of H-8/Me-19 and Me-18, and H-14/H-16 and H-17,
supported the B/C and C/D trans ring junction pattern; and the NOE correlations of Me-18/H-15b,
H-15a/H-16 and H-17, and H-17/Me-21, suggested the α-orientation of Me-21 (see Figure 3). Therefore,
compound 2 was identified as 1β,2β,3β,4β,5β,6β,7α,23ξ,26-nonahydroxyfurost-20(22),25(27)-dien-26-O-β-
D-glucopyranoside.
1
1
Figure 3. Key HMBC, H- H COSY and NOESY correlations of the compound 2.
Compound 3 was obtained as a white amorphous powder, and the molecular formula of C32
H
50
O
9
+
13
was established by the HR-ESI-MS signal at m/z 579.3590 [M + H] . The C-NMR spectrum exhibited
2 carbon signals, 27 of which were attributed to the aglycone carbons, while the remaining signals were
3

Molecules 2015, 20
13663
assignable to a characteristic of a xylosyl moiety (δ
C
104.1, 75.8, 78.9, 71.5 and 68.1), which was identified
as β-D-xylose by the coupling constant analysis of the anomeric proton (J = 7.2 Hz), the acid hydrolysis
procedure, TLC comparison, and the optical rotation determination. Among the aglycone carbon signals,
the quaternary carbon signal at δ
characteristic signal of spirostanol or norspirostanol saponin [14]. In HMBC spectrum, the anomeric
proton [4.81 (1H, d, J = 7.2 Hz)] of the xylose was correlated with δ 82.5, which was confirmed as C-1
82.5 (C-1), H- H COSY correlations of H-1/H-2/H-3/H-4,
C
109.9 (see, Table 1), was identified as an acetal carbon (C-22), a
C
1
1
for the HMQC correlation of δ
H
4.26 (H-1)/δ
C
and HMBC correlations of H-19/C-1, C-5, C-9 and C-10 (see, Figure 4). Moreover, HMBC correlations of
H-18/C-12, C-13, C-14 and C-17, H-21/C-17, C-20 and C-22, H-23/C-22, and H-27/C-24, C-25 and C-26,
were observed (see, Figure 4).The above data indicated the planar structure of 3 as spirost-25(27)-en-
1,3,5-trihydroxy-1-O-β-D-xyloside. In the NOESY spectrum of 3 (see, Figure 4), the NOE correlations of
Me-19/H-8, H-3/H-2a and H-4, H-2a/H-1, and H-4a/H-7a and H-9, indicated α-axial configurations of
H-1 and H-3, and β-orientation of Me-19, 1-OH, 3-OH and 5-OH, which supported the A/B cis ring
junction pattern; the NOE correlations of H-8/Me-19 and Me-18, and H-14/H-9 and H-7a, supported the
B/C and C/D trans ring junction pattern; the NOE correlations of Me-18/H-15b and H-20, H-15a/H-16
and H-17, and H-17/Me-21, suggested α-orientation of Me-21. These spectra data was almost similar
to those of (20S,22R)-1β,3β,5β-trihydroxyspirost-25(27)-en-5-O-β-D-glucopyranoside [8], expect for
the site of glycosylation. Therefore, compound 3 was elucidated as (20S, 22R)-spirost-25(27)-en-1β,3β,5β-
trihydroxy-1-O-β-D-xyloside.
1
13
Table 1. H-NMR and C-NMR spectral data of compounds 1–3.
1
2
3
Position
δc a
δ
a
(J in Hz)
δc b
δ
b
(J in Hz)
δc c
δ
c
(J in Hz)
H
H
H
1
2
3
4
77.8
4.25 (brs)
4.38 (brs)
4.70 (brs)
4.08 (brs)
-
79.1
4.29 (brs)
4.33 (brs)
4.77 (brs)
5.33 (brs)
82.5
4.26 (brs)
.53 (H-2a, ca.)
2
68.1
75.2
67.6
67.7
76.1
70.2
30.4
67.8
40.0
1
.85 (H-2b, ca.)
4.59 (brs)
2
.40 (H-4a, ca.)
2
.04 (H-4b, ca.)
5
6
87.4
24.9
78.6
74.0
-
74.7
36.3
-
1.93 (ca.), 2.80 (ca.)
5.03 (brs)
1.54 (ca.), 1.90 (ca.)
.98 (H-7a, ca.)
0
7
28.5
1.1 (ca.), 1.51 (ca.)
72.5
4.49 (brs)
29.2
1
.51 (H-7b, ca.)
1.67 (ca.)
1.15 (ca.)
-
8
9
34.4
46.6
46.2
21.9
1.59 (ca.)
1.19 (ca.)
34.8
37.8
46.3
21.9
40.0
43.8
48.9
2.62 (ca.)
2.05 (ca.)
-
35.4
46.3
44.9
22.3
1
1
1
1
1
0
1
2
3
4
-
1.41 (ca.), 1.44 (ca.)
1.61 (ca.), 1.67 (ca.)
1.14(ca.),1.38 (ca.)
39.7 1.62 (d, 12.0), 1.15 (ca.)
1.70 (d, 12.0), 1.24 (ca.) 40.5 1.73 (d, 12.5), 1.13 (ca.)
43.3
54.3
-
-
41.2
56.7
-
0.76 (ca.)
1.96 (ca.)
1.12 (ca.)
2
.48 (H-15a, ca.)
.38 (H-15b, ca.)
4.77 (q, 7.5)
2.58 (H-15a, ca.)
1.65 (H-15b, ca.)
4.87 (ca.)
2.07 (H-15a, ca.)
1.48 (H-15b, ca.)
4.62 (q, 7.2)
1
1
5
6
31.0
84.4
34.7
85.1
32.7
81.9
2

Molecules 2015, 20
13664
Table 1. Cont.
1
2
3
Position
δc a
δ
a
(J in Hz)
δc b
65.3
14.6
16.0
105.9
12.1
154.2
64.8
δ
b
(J in Hz)
δc c
63.5
17.0
14.4
42.4
15.5
109.9
33.7
δ
c
(J in Hz)
H
H
H
17
18
19
20
21
22
23
64.5
14.3
2.42 (ca.)
0.67 (s)
1.70 (s)
-
2.57 (ca.)
0.81 (s)
1.99 (s)
-
1.88 (ca.)
0.87 (s)
13.7
1.59 (s)
103.9
11.7
2.00(ca.)
1.58 (s)
-
1.74 (s)
-
1.10 (d, 8.0)
-
151.8
34.3
1.45 (ca.), 2.04 (ca.)
5.13 (dd, 6.0, 8.0)
1.81 (ca.)
2.26 (ca.)
2.74 (ca.)
-
2
.88 (H-24a, dd, 6.0, 14.3),
2
2
2
4
5
6
24.6
146.2
71.7
2.37 (ca.), 2.47 (ca.)
40.3
144.4
72.7
29.4
144.9
65.5
3
.10 (H-24b, dd, 8.0, 14.3)
-
-
4
4
.58 (d, 13.0)
.34 (d, 13.0)
4.75 (d, 13.0)
4.61 (d, 13.0)
5.47 (H-27a, s)
5.28 (H-27b, s)
5.0 (d, 7.8)
4.50 (d, 12.1)
4.07 (d, 12.1)
4.81(H-27a, s)
4.84 (H-27b, s)
4.81 (d, 7.2)
3.99 (ca.)
4.21 (ca.)
4.23 (ca.)
5
.35 (H-27a, s)
2
7
111.6
114.6
109.2
5
.04 (H-27b, s)
5.28 (d, 7.8)
3.95 (ca.)
1
'
'
'
'
97.4
76.2
78.6
71.9
104.2
75.6
80.0
72.1
104.1
75.8
78.9
71.5
2
4.12 (ca.)
3
4
4.01 (ca.)
4.36 (ca.)
4.02 (ca.)
4.27 (ca.)
3
.78 (t, 10.5),
5
'
'
78.8
62.8
4.22 (ca.)
79.0
63.2
3.96 (ca.)
68.1
-
4
.42 (dd, 4.5, 11.5)
4
.58 (dd, 2.0, 11.8),
6
4.52 (ca.), 4.21 (ca.)
-
4
.41 (dd, 5.5, 11.8)
1''
2''
3''
4''
5''
6''
103.8
75.8
78.5
71.7
78.6
62.6
4.89 (d, 7.7)
4.03 (ca.)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4.22 (ca.)
4.19 (ca.)
3.92 (ca.)
4.52 (ca.), 4.35 (ca.)
-
a
1
13
δ in pyridine-d
5
, in ppm from TMS; coupling constants (J) in Hz; H-NMR at 500 MHz and C-NMR at 125 MHz;
b
5 5
δ in pyridine-d , H-NMR at 600 MHz and C-NMR at 150 MHz; δ in pyridine-d
, 1H-NMR at 400 MHz
1
13
c
13
and C-NMR at 100 MHz.
1
1
Figure 4. Key HMBC, H- H COSY and NOESY correlations of the compound 3.
Additionally, the known furostanol saponins were identified by comparison of their spectroscopic
data with those reported in the literature as tupisteroide B (4) [15] and 5β-furost-Δ25(27)-en-
1
β,2β,3β,4β,5β,7α,22ξ,26-octaol-6-one-26-O-β-D-glucopyranoside (5) [16].

Molecules 2015, 20
13665
The cytotoxic activity of 1–4 towards the A549 and H1299 tumor cell lines was measured by the
−
1
MTT method. Compound 3 exhibited cytotoxicity against A549 cells (IC50 86.63 ± 2.33 μmol·L ) and
−1
H1299 cells (IC50 88.21 ± 1.34 μmol·L , see Tables 2 and 3). Considering 3 is a spirostanol saponin, our
results showed the cytotoxic activity of this type of steroidal saponin as mentioned in the literature [8,17–19].
Table 2. Activities of compounds 1–4 on proliferation of the H1299 cells.
Comp.
1 μM
3 μM
10 μM
30 μM
100 μM
IC50 μM
>100
1
2
3
4
1.93 ± 0.95 ** 13.50 ± 1.81 ** 14.69 ± 1.41 **
16.53 ± 1.26 **
16.17 ± 1.50 **
17.39 ± 0.73 **
13.47 ± 1.49 **
47.88 ± 1.38
16.90 ± 0.69 **
20.04 ± 1.36 **
3.95 ± 2.09 **
4.55 ± 1.10 **
4.01 ± 0.86 **
3.07 ± 0.52
5.75 ± 1.48 **
8.04 ± 1.94 **
9.26 ± 0.44 **
5.21 ± 0.28
11.50 ± 3.22 **
13.47 ± 0.61 **
11.46 ± 2.91 **
17.39 ± 1.11
>100
55.74 ± 0.87 ** 88.21 ± 1.34
26.07 ± 0.99 **
71.96 ± 2.49
>100
5
-FU
38.65 ± 1.59
The data are expressed as mean ± SD of three independent experiments (** p < 0.01 vs. control).
Table 3. Activities of compounds 1–4 on proliferation of the A549 cells.
Comp.
1 μM
3 μM
10 μM
30 μM
100 μM
IC50 μM
>100
1
2
3
4
3.75 ± 1.24 ** 11.62 ± 1.88 **
12.83 ± 2.02 **
11.07 ± 1.57 **
13.05 ± 1.75 **
7.01 ± 2.47 **
23.76 ± 1.22
14.35 ± 0.77 **
13.80 ± 2.05 **
20.60 ± 2.40 **
13.21 ± 1.40 **
42.18 ± 1.22
20.19 ± 3.63 **
23.11 ± 0.74 **
4.17 ± 1.30 **
3.95 ± 0.95 **
2.93 ± 1.18 **
6.97 ± 0.82
7.68 ± 1.27 **
7.90 ± 1.67 **
6.65 ± 0.94 **
9.03 ± 1.21
>100
56.17 ± 1.98 ** 86.63 ± 2.33
24.75 ± 1.62 **
69.24 ± 2.05
>100
5
-FU
42.78 ± 1.63
The data are expressed as mean ± SD of three independent experiments (** p < 0.01 vs. control).
3. Experimental Section
3.1. General Information
The IR spectra were recorded on a TENSOR-27 instrument (Bruker, Rheinstetten, Germany). ESI-MS
was performed on a Quattro Premier instrument (Waters, Milford, MA, USA). The HR-ESI-MS spectra
were recorded on an Agilent Technologies 6550 Q-TOF (Santa Clara, CA, USA). 1D and 2D NMR
spectra were recorded on Bruker-AVANCE 400, Bruker-AVANCE 500 and Bruker-AVANCE 600
instrument (Bruker, Rheinstetten, Germany) with TMS as an internal standard. The analytical HPLC was
performed on a Waters 2695 Separations Module coupled with a 2996 Photodiode Array Detector and a
Accurasil C18 column (4.6 mm × 250 mm, 5 mm particles, Ameritech, Chicago, IL, USA). Semipreparative
HPLC was performed on a system comprising an LC-6AD pump (Shimadzu, Kyoto, Japan) equipped with
a SPD-20A UV detector and a Ultimate XB-C18 (10 mm × 250 mm, 5 mm particles) or YMC-Pack-ODS-
A (10 mm × 250 mm, 5 mm particles). D101 was from Sunresin New Materials Co. Ltd. (Xi’an, China).
Silica gel was purchased from Qingdao Haiyang Chemical Group Corporation (Qingdao, China).
3
.2. Plant Material
The roots and rhizomes of T. chinensis Baker were collected from the Taibai region of Qinba
Mountains in Shaanxi Province, China, in August 2010, and identified by senior experimentalist Jitao

Molecules 2015, 20
13666
Wang. A voucher specimen (herbarium No. 20100816) has been deposited in the Medicinal Plants
Herbarium (MPH), Shaanxi University of Chinese Medicine, Xianyang, China.
3.3. Extraction and Isolation
The air-dried and powdered underground parts of T. chinensis (1.5 kg) were extracted with 65% EtOH
(
15 L) three times at 80 °C. The combined EtOH extracts were evaporated to 6 L, and applied to a
resin D101 column, eluting with H O, 20% EtOH, 60% EtOH, and 95% EtOH to give four fractions
Fr.1–Fr.4). Fr.3 (75 g) was subjected to column chromatography (CC) on silica gel, eluting with
2
(
gradient solvent system (CHCl
3
–MeOH–H
2
O, 100:0:0–0:50:50) to yield nine fractions (Fr.3-1–Fr.3-9).
–MeOH (100:1–50:50) as eluent to obtain
eight fractions (Fr.3-6-1–Fr.3-6-8). Fr.3-6-5 (150 mg) and Fr.3-6-7 (370 mg) were purified by HPLC
Fr.3-6 (5 g) was separated over silica gel using CHCl
3
−
1
(
YMC-Pack-ODS-A, 10 mm × 250 mm, 5 μm particles, flow rate: 1.0 mL·min ) with CH
45:55) as mobile phase to afford 1 (23 mg; t = 35 min), 2 (15 mg; t = 27 min), 3 (20 mg; t = 43 min),
= 65 min).
3
OH–H O
2
(
R
R
R
4
(27 mg; t
R
= 47 min) and 5 (1.8 mg; t
R
3
.4. 1β,2β,3β,4β,5β,26-Hexahydroxyfurost-20(22),25(27)-dien-5,26-O-β-D-glucopyranoside (1)
−
1 1
A white amorphous powder, IR (KBr) νmax: 3450, 2980, 1694, 1025, 907, 804, 772 cm . H-NMR
1
3
(
500 MHz, pyridine-d
5
) and C-NMR (125 MHz, pyridine-d
5
) spectral data, see Table 1; m/z 801.3855
−
[
M − H] (calcd. for C39 17, 801.3909).
H O
61
3.5. 1β,2β,3β,4β,5β,6β,7α,23ξ,26-Nonahydroxyfurost-20(22),25(27)-dien-26-O-β-D-glucopyranoside (2)
−1 1
A white amorphous powder, IR (KBr) νmax: 3475, 2980, 1742, 1062, 904, 804 cm . H-NMR (600 MHz,
1
3
) spectral data, see Table 1; m/z 711.3198 [M + Na]+
pyridine-d
5
) and C-NMR (150 MHz, pyridine-d
5
(
calcd. for C33
52
H O15Na, 711.3204).
3
.6. (20S,22R)-Spirost-25(27)-en-1β,3β,5β-trihydroxy-1-O-β-D-xyloside (3)
−
1
A white amorphous powder; IR (KBr) νmax: 3306, 2922, 1650, 1042, 989, 917, 892, 876 cm ;
1H-NMR (400 MHz, pyridine-d
) and C-NMR (100 MHz, pyridine-d
13
5
5
) spectral data, see Table 1; m/z
+
5
79.3590 [M + H] (calcd. for C32
H
51
9
O , 579.3633).
3
.7. Acid Hydrolysis of Compounds 1, 2, 3 and Absolute Sugar Configuration Determination
The solutions of compounds 1 (3 mg), 2 (3 mg) and 3 (5 mg) were hydrolyzed with 2N HCl (5 mL)
for 5 h at 80 °C, respectively. The reaction mixtures were concentrated and dried by N
was added and the mixtures were extracted with EtOAc (3 × 5 mL). The aqueous layers of 1 and 2 were
subjected to CC over silica gel eluted with MeCN–H O (8:1) to yield D-glucose, which was determined
by TLC comparison (MeCN–H O, 6:1) with the authentic sugar and the optical rotation determination
O). The aqueous layer of 3 was subjected to CC over silica gel eluted with
O (8:1–15:1) to yield D-xylose, which was identified by TLC comparison with the authentic
2
, and then water (5 mL)
2
2
2
0
[
α]
MeCN–H
sugar and the optical rotation determination [α]
D
+49.2 (c 0.16, H
2
2
2
D
0
+17.9 (c 0.14, H O).
2

Molecules 2015, 20
.8. Cytotoxicity Assay
The cytotoxic activity assays towards the A549 and H1299 tumor cell lines were measured by the
13667
3
4
−1
MTT method in vitro, using 5-fluorouracil as positive control. Briefly, 1 × 10 mL cells were seeded
into 96-well plates and allowed to adhere for 24 h. Compounds 1–4 were dissolved in DMSO and diluted
−
1
−1
with complete medium to five concentration levels (from 0.001 mmol·L to 0.1 mmol·L ) for inhibition
rate determination. After incubation at 37 °C for 4 h, the supernatant was removed before adding DMSO
(100 μL) to each well. 5-Fluorouracil (5-Fu) was used as positive control. The inhibition rate (IR) and IC50
were calculated. Values are mean ± SD, n = 3, ** p < 0.01 vs. DMEM control. Compound 3 exhibited
cytotoxicity against A549 cells (IC50 86.63 ± 2.33 μmol·L ) and H1299 cells (IC50 88.21 ± 1.34 μmol·L−1),
−1
−
1
while the positive control of 5-Fu exhibited cytotoxicity against A549 cells (IC50 42.78 ± 1.63 μmol·L )
−
1
and H1299 cells (IC50 38.65 ± 1.59 μmol·L ), respectively, (see Tables 2 and 3).
Supplementary Materials
1
13
IR, HR-ESI-MS, H-NMR C-NMR and 2D NMR spectra for compounds 1–3 can be accessed at:
http://www.mdpi.com/1420-3049/20/08/13659/s1.
Acknowledgments
This project was financially supported by the National Natural Science Foundations of China (grant
No. 81102805, 81373978), the Open Projects Program of the Key Laboratory of Tibetan Medicine
Research, Chinese Academy of Sciences (grant No. 2014-TMR-01), the Innovative Research Team in
TCM Material Foundation and Key Preparation Technology (grant No. 2012KCT-20), the Innovation
Projects of Science and Technology in Shaanxi Province (grant No. 2011KTCQ03-02, 2013KTCQ03-14),
and the Key Program of Shaanxi University of Chinese Medicine (grant No. 2015PY09).
Author Contributions
Every author has participated in the research and did his or her individual contribution to the article:
Y.L. and X.W. conducted the experiments and collected the data; D.Z. and Y.J. planned and coordinated
the experiments; H.H. and F.W. carried out the cytotoxicity biology experiments; X.Y. and Z.T. analysed
the data; X.S. designed the study, and Z.Y. planned and oversaw the research project and drafted the
paper. Finally, All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1
.
Song, X.; Liu, H. Research and Application of “Qi-Medicines” in Taibai Mountains; People’s Medical
Publishing House: Beijing, China, 2011.

Molecules 2015, 20
13668
2
.
Wu, X.; Fan, J.; Ouyang, Z.; Ning, R.; Guo, W.; Shen, Y.; Sun, Y.; Xu, Q. Tupistra chinensis
extract attenuates murine fulminant hepatitis with multiple targets against activated T lymphocytes.
J. Pharm. Pharmacol. 2014, 66, 453–465.
3
.
.
Pan, Z.; Li, Y.; Liu, J.; Ning, D.; Li, D.; Wu, X.; Wen, Y. A cytotoxic cardenolide and a saponin
from the rhizomes of Tupistra chinensis. Fitoterapia 2012, 83, 1489–1493.
4
Cai, J.; Zhu, Z.; Yu, C.; Lei, L.; Wu, S. Saponin from Tupistra chinensis Baker inhibits mouse
sarcoma S-180 cell proliferation in vitro and implanted solid tumor growth in mice. J. South. Med. Univ.
2
007, 27, 188–190.
5.
6.
7.
8.
Li, Q.; Zou, K.; Wang, Y. Experimental study on vitro anti-tumor action of “Kai-Kou-Jian” extract.
Chin. J. Ethnomed. Ethnopharm. 2007, 86, 164–167.
Liu, C.; Guo, Z.; Xue, Y.; Cheng, J.; Huang, N.; Zhou, Y.; Cheng, F.; Zou, K. Five new furostanol
saponins from the rhizomes of Tupistra chinensis. Fitoterapia 2012, 83, 323–328.
Huang, W.; Zhang, H.; Zou, K.; Chen, J.; Li, X.; Liu, C.; Huang, N. Total saponins of Tupistra chinensis
induces apoptosis in A549 cells. Neoplasma 2012, 59, 613–621.
Song, X.; Li, Y.; Zhang, D.; Jiang, Y.; Wang, W.; Song, B.; Tang, Z.; Cui, J.; Yue, Z. Two new
spirostanol saponins from the the roots and rhizomes of Tupistra chinensis. Phytochem. Lett. 2015,
1
3, 6–10.
9
.
Chai, J.; Song, X.; Wang, X.; Mei, Q.; Li, Z.; Cui, J.; Tang, Z.; Yue, Z. Two new compounds from
the roots and rhizomes of Trillium tschonoskii. Phytochem. Lett. 2014, 10, 113–117.
1
0. Li, H.; Pan, X.; Mei, Q.; Song, X.; Pei, Y.; Yue, Z. Isolation and identification of chemical
constituents from root and rhizoma of Trillium tschonoskii Maxim. J. Shenyang Pharm. Univ. 2013,
3
0, 509–516.
1
1
1
1
1
1. Yue, Z.; Qin, H.; Li, Y.; Sun, Y.; Wang, Z.; Yang, T.; Liu, L.; Wang, M.; Feng, F.; Mei, Q. Chemical
constituents of the root of Jasminum giraldii. Molecules 2013, 18, 4766–4775.
2. Hudson, C.; Dale, J. Studies on the forms of D-glucose and their mutarotation. J. Am. Chem. Soc.
1
917, 39, 320–328.
3. Zhang, X.; Chen, C.; Yang, J.; Ni, W.; Liu, H. New minor spirostane glycosides from
Ypsilandra thibetica. Helv. Chim. Acta 2012, 95, 1087–1093.
4. Yan, W.; Ohtani, K.; Kasai, R.; Yamasaki, K. Steroidal saponins from fruits of Tribulus terrestris.
Phytochemistry 1996, 42, 1417–1422.
5. Liu, C.; Guo, Z.; Xue, Y.; Zhang, H.; Zhang, H.; Zou, K.; Huang, N. Tupisteroide A–C, three new
polyhydroxylated steroidal constituents from the roots of Tupistra chinensis. Magn. Reson. Chem.
2
012, 50, 320–324.
1
6. Xu, L.; Zou, K.; Wang, J.; Wu, J.; Zhou, Y.; Dan, F.; Yang, J. New polyhydroxylated furostanol
saponins with inhibitory action against NO production from Tupistra chinensis Rhizomes. Molecules
2
007, 12, 2029–2037.
1
7. Li, Y.; Fan, L.; Sun, Y.; Miao, X.; Zhang, F.; Meng, J.; Han, J.; Zhang, D.; Zhang, R.; Yue, Z.; et al.
Paris saponin VII from trillium tschonoskii reverses multidrug resistance of adriamycin-resistant
MCF-7/ADR cells via P-glycoprotein inhibition and apoptosis augmentation. J. Ethnopharmcol.
2
014, 154, 728–734.

Molecules 2015, 20
13669
1
1
8. Li, Y.; Sun, Y.; Fan, L.; Zhang, F.; Meng, J.; Han, J.; Guo, X.; Zhang, D.; Zhang, R.; Yue, Z.; et al.
Paris saponin VII inhibits growth of colorectal cancer cells through Ras signaling pathway.
Biochem. Pharmacol. 2014, 88, 150–157.
9. Pan, W.; Chang, F.; Wei, L.; Wu, Y. New flavans, spirostanol sapogenins, and a pregnane genin from
Tupistra chinensis and their cytotoxicity. J. Nat. Prod. 2003, 66, 161–168.
Sample Availability: Samples of the compounds 3–5 are available from the authors.
©
2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/4.0/).
(