Steroidal constituents from roots and rhizomes of Smilacina japonica

Article abstract of DOI:10.3390/molecules23040798

Four new steroidal constituents (1–4) along with two known steroidal glycosides (5 and 6) were isolated from the roots and rhizomes of Smilacina japonica. Analysis of their physicochemical properties and spectroscopic profiles identified the compounds as

Full text of DOI:10.3390/molecules23040798

molecules
Article
Steroidal Constituents from Roots and Rhizomes of
Smilacina japonica
Yuwen Cui 1,2,†, Xinjie Yang 3,†, Dongdong Zhang 4, Yuze Li 5, Li Zhang 3, Bei Song 5,
Zhenggang Yue 3, Xiaomei Song 3 and Haifeng Tang 1,
*
1
Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China;
polaris_101025@163.com
2
3
Department of Pharmacy, Xi’an Medical University, Xi’an 710021, China
Shaanxi Collaborative Innovation Center of Chinese Medicinal Resource Industrialization,
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; xxx211xxx@126.com (X.Y.); zhangl123666@163.com (L.Z.);
liuxingjian1981@163.com (Z.Y.); songxiaom@126.com (X.S.)
The College of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 200001, China;
zhangnatprod@163.com
The College of Life Sciences, Northwest University, Xi’an 710069, China; lyz1990yeah@163.com (Y.L.);
songbei168@126.com (B.S.)
4
5
*
Correspondence: tanghf71@fmmu.edu.cn; Tel.: +86-29-8477-4748
†
The authors contribute equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 6 March 2018; Accepted: 28 March 2018; Published: 30 March 2018
Abstract: Four new steroidal constituents (
and ) were isolated from the roots and rhizomes of Smilacina japonica.
of their physicochemical properties and spectroscopic profiles identified the compounds
as (25S)-5 -spirostan-9(11)-en-3 , 17 -diol ( ); (25S)-5 -spirostan-9(11)-en-3 , 12 -diol ( );
(25S)-5 -spirostan-9(11)-en-3 , 17 -diol-3-O- -D-glucopyranoside ( ); (25S)-5 -spirostan-9(11)-en-3
17 -diol-3-O- -D-glucopyranosyl-(1 2)-[ -D-glucopyranosyl-(1 3)]- -D-galactopyranoside (
japonicoside B ( ); and japonicoside C (
1–
4
) along with two known steroidal glycosides
(
5
6
Analysis
α
β
α
1
α
β
β
2
α
β
α
β
3
α
β,
α
β
→
β
→
β
4
);
5
6). All six compounds showed cytotoxic activity against
SMMC-7712, Bel-7402, A549, H460, and K562 human cancer cells.
Keywords: Smilacina japonica; steroidal constituents; structure identification; cytotoxicity
1. Introduction
Smilacina japonica A. Gray is a perennial herb of the genus Smilacina (Liliaceae) that is mainly
distributed in Asia, America, and Europe. As one of about 16 Smilacina species in China, S. japonica is
widely distributed in Hebei, Shanxi, Shaanxi, Gansu, and Henan Provinces and is used in traditional
Chinese medicine or consumed as a vegetable. Its roots and rhizomes are known to dispel pathogenic
wind, remove dampness, promote blood circulation, and alleviate pain, and have long been used in folk
medicine for the treatment of rheumatism, nervous headache, mastitis, carbuncle, and bruises [
Previous investigations of the bioactive compounds of S. japonica revealed four steroidal saponins [
1
6
–
–
5
8
].
].
As part of our exploration of the diversity of bioactive compounds in medicinal herbs growing in the
Qinba Mountains [9–15], we investigated the chemical constituents of S. japonica in the present study.
We identified six steroidal constituents (Figure 1), characterized their structure and pharmacological
profiles, and evaluated their cytotoxicity against various human cancer cell lines.
Molecules 2018, 23, 798; doi:10.3390/molecules23040798
www.mdpi.com/journal/molecules

Molecules 2018, 23, 798
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Figure 1. Chemical structures of compounds 1–6.
2. Results and Discussion
Compound
1 was isolated as a white, amorphous powder and was positive to
Liebermann-Burchard and Molisch chemical reactions. The HR-ESI-MS analysis revealed a positive
molecular ion peak at m/z 431.3154 [M + H]+, corresponding to the molecular formula C27H42O4
1
(calculated for 431.3161 [M + H]+). The proton H-NMR spectrum of
1 showed the presence of
four methyl protons at δH 0.92 (3H, s, H-18), 0.98 (3H, s, H-19), 1.21 (3H, d, J = 7.1 Hz, H-21), and
1.05 (3H, d, J = 7.1 Hz, H-27) and an exocyclic olefinic proton at δH 5.51 (1H, d, J = 5.3 Hz, H-11).
The 13C-NMR spectrum showed 27 carbon signals corresponding to one double-bond carbon at
δC 145.5 (C-9) and 116.2 (C-11) and four methyl groups at δC 16.4 (C-18), 17.4 (C-19), 8.2 (C-21),
and 15.4 (C-27). The quaternary carbon signal at δC 110.5 was identified as an acetal carbon (C-22),
which is characteristic of spirostanol saponin. The 13C signals of carbons near C-17, C-12 (δC 32.8),
C-13 (δC 42.9), C-14 (δC 50.5), C-16 (δC 90.1), and C-21 (δC 8.2) and the further long-range correlation
from 21-CH3 to the quaternary C-atom at δC 88.9 (C-17) was also observed, suggesting the presence of
a hydroxyl group at C-17.
The above observations were supported by 2D-NMR analysis. The proton and protonated
carbon resonances in the NMR spectrum of
1 were unambiguously assigned by the heteronuclear
single quantum correlation (HSQC) experiment. Heteronuclear multiple bond correlations (HMBCs)
of H-19/C-1, C-5, C-9, and C-10; H-4/C-2 and C-5; H-6/C-5, C-7, and C-8; H-8/C-9, C-11, and
C-14; H-18/C-12, C-13, C-14, and C-17; H-15/H-14, H-16, and H-17; H-16/C-15, C-17, and C-22;
H-21/C-17, C-20, and C-22; and H-27/C-24, C-25, and C-26 revealed the planar structure of
as spirostan-9(11)-en-3,17-diol (Figure 2). Meanwhile, in the nuclear Overhauser effect (NOE)
spectrum, NOE correlations between H19/H-4b and H-8, H-4a/H-3, and H-3/H-5 indicated an -axial
configuration of H-3 and H-5 and orientation of H-8, H-19, and 3-OH, with an A/B trans ring junction
pattern; whereas NOE correlations of H-19/H-8, H-8/H-18, H-18/H-15b, and H-20 and of H-15a/H-14
and H-16 suggested -axial configurations for H-21 and 17-OH (D/E cis ring, Figure 3), orientation
1
α
β
α
β
for H-18, and B/C and C/D trans ring junction patterns. Finally, the absolute configuration of C-25
was deduced as S based on the difference in chemical shifts between equatorial (δH 3.24) and axial
(
δH 4.03) H-26, which is usually ∆δ > 0.57 and <0.48 ppm for 25S and 25R compounds, respectively [16].
Compound 1 was identified as (25S)-5α-spirostan-9(11)-en-3β, 17α-diol (1; Figure 1).

Molecules 2018, 23, 798
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Figure 2. Key HMBC correlations of compounds 1–4.
Figure 3. Key NOESY correlations of compounds 1–4.
Compound
2 was isolated as a white, amorphous powder and was positive to
Liebermann-Burchard and Molisch chemical reactions. The HR-ESI-MS spectrum showed a positive
molecular ion peak at m/z 431.3152 [M + H]+ corresponding to a molecular formula of C27H42O4
(calculated for 431.3161 [M + H]+). The 1H-NMR spectrum of
2 showed the presence of four methyl
protons at δH 1.09 (3H, s, H-18), 0.99 (3H, s, H-19), 1.42 (3H, d, J = 6.9 Hz, H-21), and 1.08 (3H, d,
J = 6.9 Hz, H-27) and an exocyclic olefinic proton at δH 5.59 (1H, broad singlet, H-11). The 13C-NMR
spectrum showed 27 carbon signals—including one double-bond carbon at δC 148.5 (C-9) and 123.9
(C-11); four methyl groups at δC 10.9 (C-18), 18.0 (C-19), 14.0 (C-21), and 16.3 (C-27); and an quaternary
carbon signal at δC 110.1 that was identified as an acetal carbon (C-22). A comparison of proton and
carbon data showed that the resonances of
of 17-OH and appearance of 12-OH in the former. Moreover, the down-field shifted methylene signal
at δC 123.9 (C-11) and the up-field Me-18 signal at δC 10.9 suggested the existence of one -oriented
hydroxyl group at C-12 (δC 78.6), which was further confirmed by ROESY correlations between H-12
and H-14, H-14 and H-15, H-15 and H-16. The chemical structure of was supported by 2D-NMR data
2 were similar to those of 1 (Table 1), except for the absence
β
2
from HSQC, HMBC (Figure 2), and NOE spectroscopy (NOESY) (Figure 3) experiments. Compound
2
was identified as (25S)-5α-spirostan-9(11)-en-3β, 12β-diol (2; Figure 1).

Molecules 2018, 23, 798
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1
Table 1. H-NMR and 13C-NMR data for compounds 1–4 †.
1
2
3
4
No.
δH
δC
δH
δC
δH
δC
δH
δC
1.44, 1H, ca.
1.75, 1H, ca.
1.73, 1H, ca.
2.06, 1H, ca.
3.84, 1H, ca.
1.51, 1H, ca.
1.76, 1H, ca.
1.25, 1H, ca.
1.22, 1H, ca.
1.38, 1H, ca.
0.95, 1H, ca.
1.75, 1H, ca.
2.24, 1H, ca.
−
1.46, 1H, ca.
1.66, 1H, ca.
1.67, 1H, ca.
2.19, 1H, ca.
3.86, 1H, ca.
1.53, 1H, ca.
1.84, 1H, ca.
1.23, 1H, ca.
1.24, 1H, ca.
1.28, 1H, ca.
0.94, 1H, ca.
1.81, 1H, ca.
2.18, 1H, ca.
−
1.26, 1H, ca.
1.61, 1H, ca.
1.73, 1H, ca.
2.06, 1H, ca.
3.97, 1H, ca.
1.36, 1H, ca.
1.87, 1H, ca.
1.03, 1H, ca.
1.16, 1H, ca.
1.25, 1H, ca.
0.92, 1H, ca.
1.73, 1H, ca.
2.09, 1H, ca.
−
1.25, 1H, ca.
1.61, 1H, ca.
1.73, 1H, ca.
2.06, 1H, ca.
3.94, 1H, ca.
1.36, 1H, ca.
1.88, 1H, ca.
1.06, 1H, ca.
1.18, 1H, ca.
1.24, 1H, ca.
0.93, 1H, ca.
1.73, 1H, ca.
2.08, 1H, ca.
−
1
35.3
36.1
35.3
35.3
2
3
31.7
69.6
32.9
70.4
29.5
76.4
29.5
76.7
4
5
34.4
42.9
39.3
43.7
34.4
42.6
34.4
42.7
6
28.1
28.9
28.2
28.2
7
33
33.5
33
33
8
9
10
36.1
145.5
37.5
36.3
148.5
38.2
36.2
145.5
37.6
36.2
145.5
37.6
−
−
−
−
5.51, 1H, d,
J = 5.3 Hz
1.78, 1H, ca.
3.07, 1H, d,
J = 17.4 Hz
−
5.45, 1H, d,
J = 5.3 Hz
1.77, 1H, ca.
3.06, 1H, d,
J = 17.4 Hz
−
5.45, 1H, d,
J = 5.3 Hz
1.78, 1H, ca.
3.06, 1H, d,
J = 17.4 Hz
−
11
116.2
5.59, 1H, brs
123.9
116.4
116.4
4.33, 1H, brs
12
32.8
78.6
33
33
13
14
42.9
50.5
−
45.2
53.2
43.1
50.6
43.1
50.6
2.13, 1H, ca.
1.51, 1H, ca.
2.32, 1H, ca.
4.45, 1H, d,
J = 7.2 Hz
1.48, 1H, ca.
1.52, 1H, ca.
2.07, 1H, ca.
2.13, 1H, ca.
1.53, 1H, ca.
2.32, 1H, ca.
4.48, 1H, d,
J = 7.2 Hz
2.13, 1H, ca.
1.52, 1H, ca.
2.31, 1H, ca.
4.45, 1H, d,
J = 7.2 Hz
15
16
17
31.9
90.1
88.9
32.4
81.5
61.9
32.1
90.3
89.1
32.1
90.3
89.1
4.62, 1H, ca.
2.34, 1H, t,
J = 6.0 Hz
0.1.09, 3H, s
0.99, 3H, s
2.14, 1H, ca.
1.42, 3H, d,
J = 6.9 Hz
−
−
−
−
18
19
20
0.92, 3H, s
0.98, 3H, s
2.23, 1H, ca.
1.21, 3H, d,
J = 7.1 Hz
−
16.4
17.4
44.8
10.9
18.0
43.8
0.92, 3H, s
0.68, 3H, s
2.21, 1H, ca.
1.23, 3H, d,
J = 7.1 Hz
−
16.6
17.4
45.1
0.92, 3H, s
0.84, 3H, s
2.22, 1H, ca.
1.22, 3H, d,
J = 7.1 Hz
−
16.6
17.4
45.1
21
22
23
8.2
110.5
25.6
14.0
110.1
26.4
8.5
109.8
25.8
8.5
109.8
25.8
1.45, 1H, ca.
1.92, 1H, ca.
1.32, 1H, ca.
2.06, 1H, ca.
1.55, 1H, ca.
3.24, 1H, d,
J = 11.0 Hz
1.47, 1H, ca.
1.92, 1H, ca.
1.34, 1H, ca.
2.04, 1H, ca.
1.59, 1H, ca.
3.33, 1H, d,
J = 11.0 Hz
4.09, 1H, dd,
J = 2.4, 11.0
Hz
1.46, 1H, ca.
1.92, 1H, ca.
1.33, 1H, ca.
2.05, 1H, ca.
1.55, 1H, ca.
3.24, 1H, d,
J = 11.0 Hz
1.46, 1H, ca.
1.91, 1H, ca.
1.32, 1H, ca.
2.03, 1H, ca.
1.54, 1H, ca.
3.24, 1H, d,
J = 11.0 Hz
24
25
24.8
26.5
26.2
27.6
25.1
26.3
25.1
26.7
26
64.2
65.2
64.4
64.4
4.04, 1H, dd,
J = 2.4, 11.0 Hz
4.03, 1H, dd,
J = 2.4, 11.0 Hz
4.04, 1H, dd,
J = 2.4, 11.0 Hz
1.05, 3H, d,
J = 7.1 Hz
1.08, 3H, d,
J = 6.9 Hz
1.05, 3H, d,
J = 7.1 Hz
1.06, 3H, d,
J = 7.1 Hz
27
Sugar
Gal-1
2
15.4
16.3
15.6
15.6
4.98, 1H, d,
J = 7.6 Hz
4.47, 1H, ca.
4.18, 1H, t,
J = 6.0 Hz
4.61, 1H, d,
J = 3.0 Hz
4.24, 1H, dd,
J = 3.0, 9.4 Hz
4.48, 2H, ca.
4.91, 1H, d,
J = 7.6 Hz
4.62, 1H, ca.
102.2
72.1
76.4
101.8
80.5
85.5
3
4.18, 1H, ca.
4.53, 1H, ca.
4.01, 1H, ca.
4
5
6
69.8
72.7
77.6
74.9
62
4.26, 1H, ca.
4.80, 1H, ca.
5.16, 1H, d,
J = 7.5 Hz
4.13, 1H, ca.
3.81, 1H, ca.
3.99, 1H, ca.
4.07, 1H, ca.
4.02, 1H, ca.
4.65, 1H, ca.
5.27, 1H, d,
J = 7.5 Hz
4.06, 1H, ca.
3.97, 1H, ca.
4.26, 1H, ca.
4.12, 1H, ca.
4.37, 1H, ca.
4.59, 1H, ca.
59.9
0
Glc-1
104.6
0
2
3
4
5
75
0
78.3
71.2
76.2
0
0
0
60.9
6
00
Glc-1
106.4
00
2
3
4
5
74.5
77.9
69.6
77
00
00
00
00
62.6
6
†
Assignments aided by the HSQC, HMBC, and NOESY experiments; 1H- and 13C-NMR were measured at 400 and
100 MHz in pyridine-d5.

Molecules 2018, 23, 798
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Compound
3
was isolated as a white, amorphous powder and was positive to
Liebermann-Burchard and Molisch chemical reactions. The HR-ESI-MS spectrum showed a negative
molecular ion peak at m/z 591.3508 [M + H]− corresponding to a molecular formula of C33H52O9
(calculated for 591.3533 [M + H]−). The 1H-NMR spectrum of
3 revealed the presence of four methyl
protons at δH 0.92 (3H, s, H-18), 0.68 (3H, s, H-19), 1.22 (3H, d, J = 7.1 Hz, H-21), and 1.05 (3H, d,
J = 7.1 Hz, H-27) and an anomeric proton at δH 4.98 (1H, d, J = 7.6 Hz, H-Glc-1). The 13C-NMR spectrum
showed 33 carbon signals; one double-bond carbon at δC 145.5 (C-9) and 116.4 (C-11) and four methyl
groups at δC 16.6 (C-18), 17.4 (C-19), 8.5 (C-21), and 15.6 (C-27), as well as an anomeric carbon at δC
102.2 (C-Glc-1). In addition, the quaternary carbon signal at δC 109.8 was identified as an acetal carbon
(C-22), which is characteristic of spirostanol saponin. A comparison of the proton and carbon data with
those of compound
linked to the C-3 of the aglycone (C3 + 6.8 ppm). Acid hydrolysis of
confirmed by GC analysis of the trimethylsilyl-l-cysteine derivatives of compound
and authentic sugars. The large J values (J >7.0 Hz) of the anomeric proton signals reflected the
configuration of D-glucose. Compound was identified as (25S)-5 -spirostan-9(11)-en-3 , 17 -diol
3-O- -D-glucopyranoside ( ; Figure 1) based on HSQC, HMBC (Figure 2), and NOESY (Figure 3) data.
Compound was isolated as a white, amorphous powder and was positive to
1
(Table 1) indicated that a glucose unit (102.2, 72.1, 76.4, 69.8, 74.9, and 62.0) was
yielded D-glucose, which was
hydrolysate
3
3
β
3
α
β
α
β
3
4
Liebermann-Burchard and Molisch chemical reactions. The HR-ESI-MS spectrum showed
a negative molecular ion peak at m/z 915.4565 [M + H]−, corresponding to a molecular
formula of C45H72O19 (calculated for 915.4590 [M + H]−).
NMR spectra for
was replaced by -D-glucopyranosyl-(1
-D-galactopyranoside in 4. The HMBC spectrum of
4
and
→
1
2)-
showed similar features, except that the 3-OH in
-D-glucopyranosyl-(1 3)]-
1
β
[
β
→
β
4
(Figure 2)
revealed correlations between H-Glc-100/C-Gal-3, H-Glc-10/C-Gal-2, and H-Gal-1/C-3, with the
two glucose units linked to C-Gal-2 and C-Gal-3 of the inner galactose unit, which was
itself linked to C-3 of aglycone. This was confirmed by acid hydrolysis and subsequent
GC analysis of the hydrolysates and 2D-NMR analysis. Coupling constants of the anomeric
proton signals suggested
β-configuration of D-glucose and D-galactose. Based on this evidence,
compound was identified as (25S)-5
4
α
-spirostan-9(11)-en-3 , 17 -diol 3-O- -D-glucopyranosyl-(1 2)-
β
α
β
→
[β-D-glucopyranosyl-(1→3)]-β-D-galactopyranoside (4; Figure 1).
The two known glycosides were identified as japonicoside
B
(
5
,
Figure 1) and
japonicoside C ( -D-glucopyranosyl-(1 2)-
-D-xylopyranosyl-(1 -D-galactopyranoside ( ), (25S)-5 -spirostan-
9(11)-en-3 , 17 , 24 -diol 3-O- 2)-[ -D-xylopyranosyl-(1 3)]- -D-glucopyranosyl
(1 4)- -D-galactopyranoside (6), respectively, by comparing their spectroscopic data (Supplementary
6
, Figure 1); (25S)-5
3)]- -D-glucopyranosyl(1
-D-glucopyranosyl-(1
α
-spirostan-9(11)-en-3
β, 17
α
-diol 3-O-
β
→
[β
→
β
→
4)-
→
β
5
α
β
α
α
β
β
→
β
→
β
Materials) with those reported in the literature [6].
Because steroidal saponins have been reported to possess varying cytotoxic activity against
various cancer cell lines [17
Bel-7402, A549, H460, and K562 tumor cells of compounds
all the compounds exhibited cytotoxicity with the cell lines. Compound
antitumor effect than : it seemed that the presence of a free hydroxyl group at C-12 was more potent
than a free hydroxyl group at C-17. Compounds shared the same aglycone, but exhibited
–
19], in this paper, the cytotoxic activity against human SMMC-7712,
were evaluated by MTT method and
exhibited a more potent
1–6
2
1
1, 3–5
different activities. This suggested that the structural differences such as the category, the number, and
the sequence of the oligosaccharide chain at C-3 played a role in terms of antitumor effect. Meanwhile,
compared with
have no differences. It seemed that the presence of a free hydroxyl group at C-24 has less effect on
the cytotoxic activity. Furthermore, compounds and exhibited moderate cytotoxicity against A549
cells with IC50 values of 14.4 M and 12.3 M, respectively, while compounds and displayed
no activity (IC50 > 100 M). It seemed that the differences of the composition of the oligosaccharide
5, compound 6 has one more free hydroxyl group than 5, but their cytotoxic activity
2
4
µ
µ
1
,
3,
5
6
µ
chain have an effect on the cytotoxic activity and selectivity of the various cell lines. Thus, our results
indicated that the antitumor effects of steroidal constituents from this species are very sensitive to their

Molecules 2018, 23, 798
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precise functionalization. Therefore, more extensive studies are needed before a clear structure-activity
relationship can be reached.
3. Materials and Methods
3.1. Materials
Optical rotations were recorded in MeOH using a Rudolph research analytical automatic
polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). Infrared (IR) spectra were
recorded on a TENSOR-27 instrument (Bruker, Billerica, MA, USA). High-resolution electrospray
ionization mass spectrometry (HR-ESI-MS) analysis was carried out on a model 6550 quadrupole
time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). One-dimensional
(1D) and 2D nuclear magnetic resonance (NMR) spectra were recorded on an AVANCE400
instrument (Bruker) with tetramethylsilane as an internal standard. High-performance liquid
chromatography (HPLC) was performed on a 2695 Separations Module (Waters, Milford, MA, USA)
coupled with a 2996 Photodiode Array Detector and octadecylsilyl (ODS)-3 column (4.6
5 mm particles; COSMOSIL, Tokyo, Japan). Semi-preparative HPLC was performed on a LC-6AD
pump equipped with a SPD-20A ultraviolet detector and an Ultimate XB-C18 column (10 250 mm,
×
250 mm,
×
5 mm particles) (Shimadzu, Kyoto, Japan). Gas chromatography (GC) was performed on an 7890 A
chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with an HP-5 capillary column
(30 m × 320 mm × 0.25 mm; Agilent Technologies). Sephadex LH-20 and C-18 (40–75 mm) silica gels
were purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden), and silica gel was also
purchased from Qingdao Haiyang Chemical Group Corporation (Qingdao, China).
3.2. Plant Material
Smilacina japonica A. Gray was collected in August of 2014 from the Taibai region of the Qinba
Mountains in the Shaanxi Province of China and was authenticated by Professor Jitao Wang (Shaanxi
University of Chinese Medicine). A voucher specimen (herbarium no. 20140826) has been deposited in
the Medicinal Plants Herbarium, Shaanxi University of Chinese Medicine (Xianyang, China).
3.3. Extraction and Isolation
The air-dried powder of S. japonica rhizomes and roots of (6.3 kg) was extracted three times with
◦
80% EtOH under reflux at 80 C. After removing the solvent, the concentrated residue was successively
partitioned with petroleum ether and n-BuOH. The n-BuOH extract (200 g) was subjected to column
chromatography (CC) on silica gel with gradient elution (CHCl3–MeOH–H2O, 100:0:0
which yielded five fractions (Fr.1–5). Fr.1 (10.5 g) was subjected to CC on silica gel; elution with
CHCl3–MeOH–H2O (100:0:0–10:1:0.1) yielded compound (8.3 mg) and compound (6.8 mg).
−
65:35:10),
1
2
Fr.4 (22 g) was subjected to CC on silica gel; elution with CHCl3–MeOH–H2O (100:0:0–70:30:5) yielded
four subfractions (Fr.4-1–4-4). Fr.4-1 (1.6 g) was subjected to CC on ODS gel; elution with MeOH-H2O
(10:90–70:30) yielded compound
3 (15.5 mg). Fr.4-3 (3.4 g) was subjected to CC on ODS gel; elution with
MeOH–H2O (10:90–70:30) yielded three subfractions (Fr.4-3-1–4-3-3). Fr.4-3-2 (188 mg) was purified by
HPLC (flow rate: 1.5 mL/min) with MeCN–H2O (46:54) as the mobile phase, yielding compound
4
6
(18.6 mg; retention time [tR] = 41.2 min), compound
(11.6 mg; tR = 31.2 min).
5 (37.6 mg; tR = 33.4 min), and compound
3.4. (25S)-5α-Spirostan-9(11)-en-3β,17α-diol (Compound 1)
This compound was a white amorphous powder (purity >98%) with the following spectral
features. [α]D23.1
−
31.5 (c 1.57, MeOH); IR (KBr)νmax: 3407, 2934, 1653, 1072, 1035, 979, 913, 892,
1
845 cm−1; H- and 13C-NMR spectral data (Table 1): positive HR-ESI-MS m/z 431.3154 [M + H]+
(calculated for C27H43O4: 431.3161 [M + H]+).

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3.5. (25S)-5α-Spirostan-9(11)-en-3β,12β-diol (Compound 2)
This compound was a white amorphous powder (purity of 94%) with the following spectral
features. [α]D23.3
−
39.0 (c 0.61, MeOH); IR (KBr)νmax: 3407, 2929, 1664, 1070, 1037, 979, 915, 893,
1
847 cm−1; H- and 13C-NMR spectral data (Table 1): positive HR-ESI-MS m/z 431.3152 [M + H]+
(calculated for C27H43O4: 431.3161 [M + H]+).
3.6. (25S)-5α-Spirostan-9(11)-en-3β,17α-diol 3-O-β-D-glucopyranoside (Compound 3)
This compound was a white amorphous powder (purity >98%) with the following spectral
features. [α]D22.9
−
96.6 (c 0.16, MeOH); IR (KBr)νmax: 3410, 2939, 1658, 1071, 1037, 978, 916, 893,
845 cm−1; 1H- and 13C-NMR spectral data (Table 1): negative HR-ESI-MS m/z 591.3508 [M
(calculated for C33H51O9: 591.3533 [M − H]−).
−
H]−
3.7. (25S)-5α-Spirostan-9(11)-en-3β,17α-diol3-O-β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-
β-D-galactopyranoside (Compound 4)
This compound was a white amorphous powder (purity >98%) with the following spectral
features. [α]D23.0
−
21.3 (c 0.70, MeOH); IR (KBr)νmax: 3410, 2930, 1652, 1072, 1035, 977, 915, 893,
847 cm−1; 1H- and 13C-NMR spectral data (Table 1): negative HR-ESI-MS m/z 915.4565 [M
(calculated for C45H71O19: 915.4590 [M − H]−).
−
H]−
3.8. Acid Hydrolysis of Compounds 3–4 and Determination of Absolute Configuration of Sugars
Compounds
3
and
4
(3 and 4 mg) were individually hydrolyzed with 1 N HCl–dioxane (1:1, 3 mL)
◦
at 60 C for 6 h. After dilution with H2O (5 mL), the reaction mixture was extracted with EtOAc,
yielding distinct EtOAc and H2O phases. The latter was evaporated under reduced pressure.
After adding H2O (5 mL), the acidic solution was evaporated again; this procedure was repeated
until a neutral solution was obtained. This was then evaporated and dried in a vacuum, yielding a
monosaccharide residue that was dissolved in pyridine (0.5 mL); 2◦mg of l-cysteine methyl ester
hydrochloride was added and the mixture was maintained at 60 C for 2 h, evaporated under
a stream of N2, and dried in a vacuum. After adding 0.2 mL of N-trimethylsilylimidazole, the mixture
◦
was maintained at 60 C for 1 h and then partitioned between n-hexane and H2O (2 mL each).
The n-hexane extract was analyzed by GC under the following conditions: HP-5 capillary column
◦
(
30 m × 320 mm × 0.25 µm); flame ionization detector; detector temperature = 280 C; injecti◦on
◦
◦
temperature = 250 C; initial temperature = 100 C for 2 min, followed by an increase to 280 C
at a rate of 10 ◦C/min; final temperature = 280 ◦C for 5 min; and N2 gas as a carrier. The absolute
configurations of sugars isolated from the hydrolysates of compounds
3 and 4 were determined
by comparing the tR of their trimethylsilyl-l-cysteine derivatives D-glucose (tR = 21.22 min) and
D-galactose (tR = 21.56 min) with those of authentic sugars prepared by a similar procedure.
3.9. Cytotoxicity Assay
The cytotoxic activity of the isolated compounds against SMMC-7712, Bel-7402, A549,
H460, and K562 human cancer cell lines was evaluated with the (3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide) assay using 5-fluorouracil as a positive control.
Briefly,
1 × 104 cells mL−1 were seeded in 96-well plates and allowed to adhere for 24 h. Compounds 1−6
were dissolved in dimethylsulfoxide (DMSO) and 6-fold dilutions were prepared in complete medium
◦
µ
mol L−1) for determination of inhibition rate. After incubation at 37.8 C for 24 h,
(from 100–0.1
the supernatant was removed and DMSO (100
µL) was added to each well. The inhibition rate and
half-maximal inhibitory concentration (IC50) were calculated. Compounds 1−6 showed cytotoxicity
against human SMMC-7712, Bel-7402, A549, H460, and K562 cell lines; the IC50 values are shown
in Table 2.

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Table 2. Cytotoxicities of compounds 1–6 against five human cancer cell lines in vitro (IC50, µM) a.
Cell Lines
A549
Compounds
SMMC-7721
Bel-7402
H460
K562
b
2.4 ± 1.9
10.4 ± 3.9
11.2 ± 4.4
8.6 ± 3.4
7.2 ± 4.6
7.4 ± 5.8
8.8 ± 5.3
4.3 ± 2.1
13.3 ± 4.6
6.9 ± 1.7
11.4 ± 4.1
4.4 ± 1.1
31.9 ± 2.1
30.3 ± 2.4
4.0 ± 1.6
>100
1.7 ± 2.8
33.7 ± 2.2
26.2 ± 2.7
31.4 ± 1.7
21.8 ± 2.1
34.4 ± 3.2
30.3 ± 3.7
1.0 ± 0.9
25.5 ± 3.5
29.3 ± 5.3
24.2 ± 2.9
>100
5-Fu
1
2
3
4
5
6
14.4 ± 2.8
>100
12.3 ± 2.5
>100
>100
15.8 ± 5.4
12.3 ± 5.5
a IC50 values are means from three independent experiments (average
±
SD) in which each compound concentration
was tested in three replicate wells; b 5-fluorouracil (5-Fu) as positive control.
Supplementary Materials: The physical and spectroscopic data of compounds
compounds 1–4 are available online.
5–
6, and NMR and MS spectra of
Acknowledgments: The research work was financially supported by the National Natural Science Foundation
of China (No. 81503195) and the Innovative Research Team in TCM Material Foundation and Key Preparation
Technology (Grant No. 2012KCT-20); and the research work was supported by the Open Research Fund of
Key Laboratory of Basic and New Herbal Medicament Research, Shaanxi University of Chinese Medicine
(No. 2017KF02,17JS030), the Natural Science Foundation of Shaanxi Provincial Department of Education (Grant
No. 16JK1219), and the Research Foundation of Shaanxi University of Chinese Medicine (Grant No. 2015PY09).
Author Contributions: H.T. conceived and designed the experiments; X.Y. and D.Z. collected the NMR data. Y.L.
contributed to the acid hydrolysis and GC analysis. L.Z. and B.S. realized the evaluation of bioactivities; Z.Y. and
X.S. analyzed the data; and Y.C. completed the isolation and structural elucidation and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–6 are available from the authors.
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2018 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
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).