Three spirostanol saponins and a flavane-O-glucoside from the fresh rhizomes of Tupistra chinensis

Article abstract of DOI:10.1016/j.fitote.2015.02.008

Four new compounds, including three new spirostanol saponins [tupistroside G-I (1-3)] and a new flavane-O-glucoside [tupichiside A (4)], together with ten known compounds, were isolated from the fresh rhizomes of Tupistra chinensis. The structures of the new compounds were elucidated by spectroscopic analysis and chemical evidence. All compounds were tested in vitro for their cytotoxic activities against the Human LoVo and BGC-823 cell lines, and six of them were found to possess potent cytotoxicity. Compounds 2, 8 and 9 showed significant cytotoxicity against the tested tumor cell lines with IC₅₀ values ranging from 0.2 to 0.9 μM.

Full text of DOI:10.1016/j.fitote.2015.02.008

Fitoterapia 102 (2015) 102–108
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Three spirostanol saponins and a flavane-O-glucoside from the
fresh rhizomes of Tupistra chinensis
Yan-Hua Xiao a,b, Hai-Long Yin c, Li Chena, Yin Tian a, Shi-Jun Liu a, Guang-Jie Zhang a,
a,
Heng-Wen Chend, Hong Jin e, Bin Li a, , Jun-Xing Dong
⁎
⁎
a
Beijing Institute of Radiation Medicine, Beijing 100850, China
Institute of Chemical Defense, Beijing 102205, China
Humanwell Healthcare Group Co., Ltd, Wuhan 430075, China
b
c
d
Guanganmen Hospital of China Academy of Chinese Medical Sciences, Beijing 100053, China
Beijing Institute of Disease Prevention and Control, 100071, China
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Four new compounds, including three new spirostanol saponins [tupistroside G-I (1–3)] and a
new flavane-O-glucoside [tupichiside A (4)], together with ten known compounds, were isolated
from the fresh rhizomes of Tupistra chinensis. The structures of the new compounds were
elucidated by spectroscopic analysis and chemical evidence. All compounds were tested in vitro
for their cytotoxic activities against the Human LoVo and BGC-823 cell lines, and six of them were
found to possess potent cytotoxicity. Compounds 2, 8 and 9 showed significant cytotoxicity
against the tested tumor cell lines with IC50 values ranging from 0.2 to 0.9 μM.
Received 24 December 2014
Accepted in revised form 12 February 2015
Accepted 13 February 2015
Available online 21 February 2015
Keywords:
Tupistra chinensis
Spirostanol saponins
Flavane-O-glucoside
Tupistroside
© 2015 Elsevier B.V. All rights reserved.
Tupichiside
Cytotoxicity
1. Introduction
As a part of a continuing study for the discovery of novel
cytotoxic agents, four new compounds, including three
Tupistra chinensis (Liliaceae), mainly distributed in south-
western China, is a rich source of steroidal sapogenins, saponins
and flavans [1–6]. These components exhibit diverse biological
activities such as anti-inflammatory, ion channel-blocking,
immune-stimulating, anti-fungal [7,8] and anti-tumor proper-
ties [9]. As a folk medicine, this species has usually been used to
reduce carbuncles and ameliorate pharyngitis [10]. In our
screening for cytotoxic agents from Chinese medicinal plants,
the ethanol extract from the fresh rhizomes of T. chinensis
showed inhibitory effect towards several human cancer cell
lines.
new spirostanol saponins [tupistroside G-I (1–3)] and
a new flavane-O-glucoside [tupichiside A (4)], together
with ten known compounds identified as isorhodeasapigenin
3-O-β-D-glucopyranoside (5), rhodeasapogenin 3-O-β-D-
glucopyranoside (6) [11], (25R)-spirostane-1β,3β,5β-triol-3-
O-β-D-glucopyranoside (7) [12], 3-O-β-D-glucopyranosyl-3-
epiruscogenin (8), 3-O-β-D-glucopyranoside-3-ruscogenin (9)
[13], (2R,3R)-3,4′-dihydroxy-7-methoxyflavane (10) [14],
tupichinol A (11), tupichinol B (12), tupichinol C (13) [3], and
(2R)-7,4′-dihydroxy-8-methylflavane (14) [15], were isolated
from the fresh rhizomes of T. chinensis (Fig. 1). All compounds
were tested for cytotoxicity against Human LoVo and BGC-823
cell lines in vitro using the MTT assay method. This paper deals
with the isolation, structure elucidation and cytotoxic activities
of these compounds.
⁎
Corresponding authors. Tel.: +86 10 66931314; fax: +86 10 68164257.
E-mail address: djx931314@163.com (J.-X. Dong).
http://dx.doi.org/10.1016/j.fitote.2015.02.008
0367-326X/© 2015 Elsevier B.V. All rights reserved.

Y.-H. Xiao et al. / Fitoterapia 102 (2015) 102–108
103
Fig. 1. Structures of compounds 1–14.
2. Experimental part
2.3. Extraction and isolation
2.1. General experimental procedures
The fresh rhizomes of T. chinensis (80 kg) were cut into
pieces and extracted with 60% EtOH for three times under
UV spectra were recorded using the UV-2500PC spectrom-
eter (Shimadzu, Japan). IR spectra were recorded on the Bio-
Rad FTS-65A spectrometer (Bio-Rad, Richmond, VA, USA).
Optical rotations were measured with a Perkin-Elmer 343
polarimeter (PerkinElmer, Waltham, MA, USA). 1H and 13C
NMR spectra were obtained on a Bruker ECA-400 MHz and
Varian UNITYINOVA 600 (Varian, Palo Alto, CA, USA), and the
chemical shifts were given on δ (ppm) scale with TMS as an
internal standard. The HR-ESI-MS were measured on a 9.4 T Q-
FT-MS Apex Qe (Bruker Co. Billerica, MA, US). Macroporous
resin AB-8 (Nan Kai College Chemical Inc., Tianjin, China), silica
gel (60–120 mesh, 200–300 mesh, Qingdao Marine Chemical
Group Co. China), Rp-C18 silica gel (100–200 mesh, YMC,
Japan) and Sephadex LH-20 (Pharmacia, Sweden) were
employed for column chromatography. TLC was carried out
using silica gel 60 (N230 mesh, Qingdao Marine Chemical
Group Co.) and GF254 plates precoated with silica gel 60. Spots
on TLC were visually observed under UV light and/or by
spraying with anisaldehyde–H2SO4 reagent followed by
heating.
reflux. After removal of the solvent in a vacuum, the
concentrated residue was partitioned with petroleum ether,
CHCl3 and n-BuOH successively. The n-BuOH phase (400 g out
of 1350 g) was dissolved in water (1.0 L), and then was
subjected to macroporous resin column chromatography,
eluting with a gradient of EtOH in water (100% water to 95%
EtOH). The 75% EtOH eluate (70 g out of 135 g) was subjected
to silica gel column chromatography (CC) eluted with CHCl3–
MeOH gradient (100% CHCl3 to 100% MeOH) to yield 110
fractions. Fractions 9–10 (31 mg) and 41–43 (85 mg) were
chromatographed by Sephadex LH-20 (with CHCl3-MeOH, 1:1)
to obtain compound 13 (25 mg) and compound 11 (65 mg)
respectively. Fraction 21–35 (6.2 g) was subjected to silica gel
CC eluted with CHCl3–MeOH gradient (from 50:1 to 10:1) to
yield twenty fractions, A1–A20. Fraction A7–A9 (87 mg) was
chromatographed by Sephadex LH-20 (with CHCl3–MeOH,
1:1) to obtain compound 10 (21 mg) and compound 14
(17 mg). Fraction A16 (31 mg) was filtered and
chromatographed by Sephadex LH-20 (with MeOH) to obtain
compound 12 (11 mg). Fraction 56–64 (4.8 g) was purified by
CC (CHCl3–MeOH, 35:1 to 5:1) to give seventeen fractions, B1–
B17. Fraction B6–B10 (245 mg) was further separated by
repeated preparative HPLC eluted with 65% methanol, giving
compound 5 (13 mg) and compound 6 (11 mg). Fraction B13
(36 mg) was purified by Rp-C18 silica gel (55% methanol) to
obtain compound 7 (19 mg). Fraction 71–80 (7.3 g) was
applied to silica gel CC eluted with CHCl3–MeOH (from 25:1 to
3:1) to give fifteen fractions, C1–C15. Fraction C11–C13
(285 mg) was further separated by repeated preparative
2.2. Plant material
The fresh rhizomes of T. chinensis were collected from
Shennongjia Forest District, Hubei province of China in May
2013 and identified by Dr. Bin Li (Beijing Institute of Radiation
Medicine). A voucher specimen (No. 2013-0501) was depos-
ited in the Herbarium of Beijing Institute of Radiation Medicine.

104
Y.-H. Xiao et al. / Fitoterapia 102 (2015) 102–108
HPLC eluted with 65% methanol to afford compound 3 (15 mg),
compound 8 (10 mg) and compound 9 (11 mg). Fraction 86–87
(185 mg) was purified by Sephadex LH-20 (with MeOH) and
preparative HPLC eluted with 60% methanol to obtain com-
pound 4 (13 mg). Fraction 91–94 (2.1 g) was subjected to silica
gel CC eluted with CHCl3–MeOH (from 15:1 to 1:1) to give
nine fractions, D1–D9. Fraction D4–D5 was repeatedly
chromatographed by Sephadex LH-20 (with MeOH) and
preparative HPLC eluted with 60% methanol to afford
compound 1 (15 mg) and compound 2 (17 mg).
(calcd for C33H53O10, 609.3633); 1H- and 13C-NMR (Pyridine-
d5) spectroscopic data, see Tables 1 and 2, respectively.
Tupichiside A (4): yellow amorphous powder; [α]2D0 −52.5
(c 0.04, MeOH); UV [MeOH] λmax (log ε) 276 (3.66), 245 (4.16)
nm, 204(4.69); IR (KBr) νmax 3396, 2921, 2849, 1646, 1513,
1494, 1468, 1419, 1218, 1165, 1129, 1076, 1046, 788 cm−1
;
HR-ESI-MS m/z 471.1629 [M + Na]+ (calcd for C23H28O9,
471.1626); 1H- and 13C-NMR (CD3OD) spectroscopic data, see
Table 3.
Tupistroside G (1): white amorphous powder; [α]2D0 −11.0
(c 0.10, Pyridine); IR (KBr) νmax 3396, 2925, 2852, 1647, 1468,
1376, 1087, 1046, 625 cm−1; HR-ESI-MS m/z 611.3789 [M
+ H]+ (calcd for C33H55O10, 611.3790); 1H- and 13C-NMR
2.4. Cell cultures
Human LoVo cell line from American Type Culture Collec-
tion (ATCC, Rockville, MD) and BGC-823 cell line from Cancer
Institute and Hospital of Chinese Academy of Medical Sciences
were cultured in Dulbecco's modified Eagle's medium (DMEM,
Gibco, USA) supplemented with 10% (v/v) fetal calf serum
(Gibco, USA), penicillin G (Macgene, China) 100 units mL−1
and streptomycin (Macgene, China) 100 μg mL−1, at 37 °C
under 5% CO2.
(Pyridine-d5) spectroscopic data, see Tables
1 and 2,
respectively.
Tupistroside H (2): white amorphous powder; [α]2D0 0.0 (c
0.10, Pyridine); IR (KBr) νmax 3428, 2953, 1637, 1457, 1362,
1272, 1164, 1073, 992, 919 cm−1; HR-ESI-MS m/z 593.3690 [M
+ H]+ (calcd for C33H53O9, 593.3684); 1H- and 13C-NMR
(Pyridine-d5) spectroscopic data, see Tables
1 and 2,
respectively.
2.5. Cell viability assay
Tupistroside I (3): white needles; [α]2D0 −65.0 (c 0.10,
Pyridine); IR (KBr) νmax 3396, 2923, 2851, 1647, 1468, 1084,
1043, 924, 894, 876 cm−1; HR-ESI-MS m/z 609.3615 [M + H]+
Cells were plated at a density of 2 × 104 cells mL−1 on 96-
well plates and treated with different concentrations of the
Table 1
1H (600 MHz) NMR data for compounds 1, 2, 3 and 7 (pyridine-d5).
Compound 1
H (J in Hz)
Compound 2
H (J in Hz)
Compound 3
H (J in Hz)
Compound 7
H (J in Hz)
δ
δ
δ
δ
1
2
4.14 (br s)
4.36 (br s)
4.15 (br s)
4.15 (br s)
2.54 (br d, 13.2)
2.04 (br d, 13.2)
4.57 (br s)
2.35 (d, 15.6)
2.21 (d, 15.6)
1.96 (m), 1.48 (m)
1.46 (m), 0.97 (m)
1.64 (m)
1.88 (br d, 12.6)
1.43 (br d, 12.6)
4.49 (br s)
2.63 (d, 14.4)
2.52 (d, 14.4)
5.63 (d, 6.0)
2.56 (br d, 13.8)
2.05 (br d, 13.8)
4.59 (d, 2.4)
2.34 (dd, 13.8, 2.4)
2.19 (d, 13.8)
1.97 (m), 1.48 (m)
2.03 (m), 1.44 (m)
1.65 (m)
2.55 (br d, 14.4)
2.01 (br d, 14.4)
4.56 (br s)
2.33 (br d, 15.6)
2.18 (br d, 15.6)
1.92 (m), 1.42 (m)
1.44 (m), 1.03 (m)
1.64 (m)
3
4
6
7
8
2.54 (m), 1.97 (m)
1.60 (m)
9
1.14 (m)
1.33 (m)
1.15 (m)
1.11 (m)
11
2.02 (dd, 9.6, 4.8)
1.37 (m)
2.85 (d, 11.4)
1.71 (m)
2.01 (dd, 9.6, 4.8)
1.37 (m)
1.99 (dd, 9.6, 4.2)
1.35 (m)
12
14
15
16
17
18
19
20
21
23
24
25
26
1.64 (m), 1.01 (m)
0.93 (m)
2.04 (m), 1.49 (m)
4.38 (d, 9.0)
1.52 (m)
0.98 (s)
1.53 (s)
2.31 (m)
1.07 (d, 6.6)
1.76 (m), 1.54 (m)
1.56 (m), 1.47 (m)
1.63 (m)
3.69 (d, 10.8)
3.64 (d, 10.8)
0.68 (d, 6.6)
1.72 (m), 1.12 (m)
0.93 (m)
1.98 (m), 1.50 (m)
4.22 (d, 8.4)
1.52 (d, 7.2)
1.02 (s)
1.27 (s)
2.30 (d, 6.9)
0.94 (d, 7.2)
1.61 (m), 1.53 (m)
1.54 (m), 1.42 (m)
1.62 (m)
3.68 (d, 9.9)
3.63 (m, 9.9)
0.68 (d, 6.6)
1.65 (m), 1.02 (m)
1.01 (m)
2.03 (m), 1.78 (m)
4.6 (d, 7.8)
1.82 (d, 9.0)
0.85 (s)
1.53 (s)
1.97 (m)
1.09 (d, 7.2)
2.23 (m), 1.46 (m)
2.71 (m), 0.98 (m)
1.66 (m), 1.01 (m)
0.98 (m)
2.05 (m), 1.42 (m)
4.55 (d, 9.0)
1.79 (d, 7.8)
0.84 (s)
1.52 (s)
1.93 (m)
1.14 (d, 7.2)
1.84 (m), 1.46 (m)
2.13 (m), 1.32 (m)
1.57 (m)
4.06 (d, 10.8)
3.36 (d, 10.8)
1.07 (d, 7.2)
4.47 (d, 12.0)
4.03 (d, 12.0)
4.81 (s), 4.78 (s)
5.00 (d, 7.8)
3.96 (dd, 10.2, 7.8)
3.98 (dd, 10.2, 7.8)
4.21 (dd, 9.0, 7.8)
4.26 (dd, 9.0, 7.8)
4.55(dd, 13.8, 6.6)
4.40 (dd, 13.8, 6.6)
27
1′
2′
3′
4′
5′
6′
5.00 (d, 7.8)
4.94 (d, 8.4)
4.99 (d, 7.8)
3.96 (dd, 9.0, 6.6)
3.97 (dd, 9.0, 6.6)
4.23 (dd, 9.6, 7.2)
4.25 (dd, 9.6, 7.2)
4.55 (dd, 11.4, 5.4)
4.41 (dd, 11.4, 5.4)
3.94 (dd, 9.0, 7.2)
3.91 (dd, 9.0, 7.2)
4.19 (dd, 9.0, 7.2)
4.20 (dd, 9.0, 7.2)
4.55 (dd, 11.4, 4.8)
4.41 (dd, 11.4, 4.8)
3.95 (dd, 8.4, 6.6)
3.98 (dd, 8.4, 6.6)
4.22 (dd, 9.0, 8.4)
4.26 (dd, 9.0, 8.4)
4.56 (d, 9.0)
4.40 (d, 9.0)

Y.-H. Xiao et al. / Fitoterapia 102 (2015) 102–108
105
Table 2
13C (150 MHz) NMR data for compounds 1, 2, 3 and 7 (pyridine-d5).
Table 4
Cytotoxic activity of compounds against LoVo and BGC-823 cell lines in vitro.
Compound 1
Compound 2
Compound 3
Compound 7
Compound
IC50 (μM)
δC
δC
δC
δC
LoVo BGC-823
⁎⁎
⁎⁎
1
2
3
4
5
6
7
8
73.0
33.1
75.4
35.4
75.2
36.7
29.0
34.7
45.7
44.0
21.6
40.5
41.2
55.4
33.3
80.9
62.7
17.0
13.5
42.2
16.8
110.6
28.3
28.2
30.7
69.6
17.3
102.9
75.4
78.7
71.4
78.5
62.7
74.0
32.5
74.8
38.9
139.8
124.3
36.8
30.7
50.7
44.3
24.3
40.7
41.0
56.2
33.4
80.7
62.7
17.0
13.3
42.1
16.6
110.6
28.1
28.0
32.3
69.6
17.3
103.0
75.0
78.5
71.7
78.7
62.9
73.0
33.2
75.4
35.4
75.2
36.7
32.2
34.9
45.6
44.0
21.5
40.7
40.0
56.2
33.0
81.7
63.0
16.6
13.6
41.9
15.0
109.5
29.0
28.9
144.4
65.0
108.7
102.9
75.5
78.9
71.4
78.4
62.7
73.1
33.1
75.4
35.4
75.2
36.7
28.9
34.9
45.6
44.0
21.5
40.7
40.0
56.2
32.2
81.2
62.8
16.6
13.6
42.5
14.9
109.7
26.4
26.2
27.6
65.1
16.3
102.9
75.5
78.9
71.4
78.4
62.7
2
4
8
9
0.267
NE
0.012
0.327
4.707
0.797
0.677
5.347
2.747
4.593
0.146
0.127
0.074
0.083
0.310
0.127
0.845
⁎⁎
⁎⁎
⁎⁎
⁎⁎
0.616
0.731
2.637
2.577
5.920
0.279
0.157
0.098
0.132
0.363
10
11
Cisplatin
NE—no effect.
9
Other inactive compounds were not listed in the table.
Values were mean SD.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1′
2′
3′
4′
5′
6′
Cisplatin, positive control.
Cell lines: LoVo colon cancer; BGC-823 stomach cancer.
⁎⁎
p b 0.01 as compared with the respective positive control value.
2.6. Statistical analysis
Values were expressed as mean
SD. Statistical analyses
were performed using the Student's t-test. Differences were
considered significant when associated with a probability of 5%
or less (p ≤ 0.05).
3. Results and discussion
Compound 1 was obtained as white amorphous pow-
der. Its molecular formula was assigned as C33H55O10 by
HR-ESI-MS m/z 611.3789 [M + H]+ (calcd for C33H55O10
,
611.3790), corresponding to the same molecular formula
(C33H55O10) as that of (25R)-spirostane-1β,3β,5β-triol-3-
O-β-D-glucopyranoside (compound 7) [12]. Comparable
13C and 1H chemical shifts for the A–D rings were observed
for 1 and 7, but with a noticeable difference for the carbons
of rings E and F, suggesting that the aglycone of 1 might be
different in stereostructure from those of 7 with respect to
the E- and F- parts (Tables 1 and 2). The proton signal for H-
27 moved upfield from δ 1.07 to δ 0.68, and the carbon
signal for C-27 downfield from δ 16.3 to δ 17.3, suggestive
of a 25S configuration in 1 [17], instead of 25R configuration as
in 7. The carbon signal for C-22 moved downfield from δ 109.7
to δ 110.6, and the other 13C chemical shifts for the F ring
downfield from δ 26.4 (C-23), δ 26.2 (C-24), δ 27.6 (C-25), δ
65.1 (C-26) to δ 28.3 (C-23), δ 28.2 (C-24), δ 30.7 (C-25), and δ
69.6 (C-26), respectively. The 13C shifts reported for the E/F
rings are in excellent agreement with those reported 22-S
configuration which has been observed before in a saponin
isolated from the tubers of Dichelostemma multiflorum [18] and
Asparagus racemosus [19]. Additionally, the chemical shifts and
coupling constants of H-26a (δ 3.64, d, J = 10.8 Hz) and H-26b
(δ 3.69, d, J = 10.8 Hz) on the F ring of 1 were different from
those (δ 3.36, d, J = 10.8 Hz for H-26a; δ 4.06, d, J = 10.8 Hz for
H-26b) on the F ring of 7. The signals of both protons at the 26
position of 1 closely appeared at δ 3.64–3.69, different from
those of 7, which indicates that compound 1 has a different
F-ring structure [20]. The phase-sensitive NOESY spectrum
provided certain information for the stereostructure as-
signments. All 1H NMR signals were assigned by the 1H–1H
COSY spectrum before inspection of the NOESY spectrum
(Table 1). The clear NOE correlations, H-14/H-17 and H-16,
test compounds for 4 days. Cytotoxicity was determined
using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) colorimetric assay [16].
After addition of 10 μL MTT solution (5 mg/mL), cells were
incubated at 37 °C for 4 h. After adding 150 μL DMSO, cells
were shaken to mix thoroughly. The absorbance of each well
was measured at 540 nm in a Multiscan photometer. The IC50
values were calculated by Origin software and listed in
Table 4.
Table 3
1H (600 MHz) and 13C (150 MHz) for compounds 4 (CD3OD).
Compound 4
δH (J in Hz)
δC
δ
H (J in Hz)
δC
2
3
4
5.02 (s)
4.22 (br s)
3.20 (dd, 16.2, 4.2)
2.77 (dd, 16.2, 4.2)
6.87 (d, 8.4)
79.8
67.5
34.4
4′
5′
6′
1″
2″
158.5
117.4
128.8
102.5
75.0
78.2
71.5
78.0
62.6
7.11 (d, 8.7)
7.45 (d, 8.7)
4.91 (d, 7.8)
3.45 (dd, 9.0, 6.0)
3.43 (dd, 9.0, 6.0)
3.38 (dd, 10.8, 7.2)
3.44 (dd, 10.8, 7.2)
3.88 (dd, 12.0, 3.6)
3.70 (dd, 12.0, 5.4)
3.78 (s)
5
6
7
8
128.2
104.7
6.52 (d, 8.4)
3″
158.3
114.3
154.1
113.0
134.9
128.8
117.4
4″
5″
6″
9
10
1′
2′
3′
7-OMe
8-Me
56.2
8.4
7.45 (d, 8.7)
7.11 (d, 8.7)
2.10 (s)

106
Y.-H. Xiao et al. / Fitoterapia 102 (2015) 102–108
Fig. 2. Key HMBC correlations of compounds 2, 3 and 4.
and H-20/Me-18, and the downfield shift for H-20, from δ
1.93 to δ 2.31, indicated the usual D/E cis-ring junction and
C-20s configuration. The H-16 proton showed an intense
NOE with 23 eq-H, giving evidence for an equatorial
position for the F-ring and the (S) configuration of C-22
(Fig. 3). In fact, the aglycone of the saponin from
A. racemosus differs from 1 only in the configuration of C-1
and C-5, which results in significantly different chemical
shifts for the carbons of the A/B rings in these two
compounds. Both 1 and 7 could presumably form from a
common intermediate which undergoes cyclisation to give an F
ring with differing stereochemistry at C-22. Maintenance of the
anomeric effect then results in the differing disposition of the C-
Compound 2 was a white amorphous powder. The
molecular formula was deduced as C33H53O9 from the [M
+ H]+ peak at m/z 593.3690 (calcd for C33H53O9, 593.3684) in
the HR-ESI-MS spectrum. All spectral properties of compound 2
were closely similar to those of 1. The major differences were
observed for their B-ring. Signals due to C-5 and C-6 in the 13
C
NMR spectrum of 1, which were observed at δ 75.2 and 36.7
respectively, were replaced by the signals assignable to the
olefinic carbons at δ 139.8 and 124.3 in 2. This was confirmed
by the long range correlations of δ 5.63 (H-6) with δ 32.5 (C-2),
38.9 (C-4) and 44.3 (C-10), δ 2.52 (H-4) and 1.97 (H-7) with δ
124.3 (C-6), δ 2.52 (H-4), 1.97 (H-7) and 1.27 (H-19) with δ
139.8 (C-5) in the HMBC spectrum (Fig. 2). Acid hydrolysis of 2
with 2 N HCl gave D-glucose which was confirmed on TCL.
Thus, compound 2 was deduced to be (22S,25S)-1β,3β,5β-
triol-spirost-5-ene-3-O-β-D-glucopyranoside (tupistroside H).
Compound 3 was obtained as white needles. The HR-ESI-
MS showed a quasi-molecular ion peak at m/z 609.3615 [M
+ H]+ (calcd for C33H53O10, 609.3633). Its 1H NMR spectrum
showed three methyl signals at δ 0.85 (3H, s), 1.09 (3H, J =
7.2 Hz) and 1.53 (3H, s), and instead of a doublet methyl proton
signal, the presence of the olefinic carbon signals at δ 144.4 and
27 methyl group. In addition, the presence of
a β-D-
glucopyranosyl moiety in 1 was readily recognized by the
appearance of an anomeric proton signal at 5.00 (d, J = 7.8 Hz),
in the 1H NMR spectrum and also by the characteristic six
signals at δ 102.9 (CH), 75.4 (CH), 78.7 (CH), 71.4 (CH), 78.5
(CH), and 62.7 (CH2) in the 13C NMR spectrum (Table 1). This
conclusion is in agreement with the sugar TCL analysis after acid
hydrolysis of 1 with 2 N HCl. In the HMBC spectrum, a cross peak
between the 1H NMR anomeric proton signal at δ 5.00 and the
carbon signal at δ 75.4 (C-3, aglycone) indicated glycosylation of
the aglycone at C-3. Compound 1 was therefore established as
(22S,25S)-1β,3β,5β-triol-spirost-3-O-β-D-glucopyranoside
(tupistroside G).
108.7 [21] and a quaternary carbon signal at δ 109.5 in the 13
C
NMR spectrum [22] indicated that 3 was a △25-spirostanol
derivative, which was confirmed by combination of 1H–1H
COSY, HSQC and HMBC spectrum. And the geminal protons at
Fig. 3. NOE correlation of 1 in pyridine-d5.

Y.-H. Xiao et al. / Fitoterapia 102 (2015) 102–108
107
C-27 were observed at δ 4.78 and 4.81 as two singlets. Acid
hydrolysis of 3 with 2 N HCl afforded D-glucose, which was
recognized by the appearance of an anomeric proton signal at δ
5.00 (1H, d, J = 7.8 Hz) in the 1H NMR spectrum and the
characteristic six carbon signals at δ 102.9 (CH), 75.5 (CH), 78.9
(CH), 71.4 (CH), 78.4 (CH), and 62.7 (CH2) in the 13C NMR
spectrum (Table 1). After having excluded signals due to the
glucose residue, the remaining carbon and proton signals
implied a spirostanetriol moiety due to the presence of two
oxymethine [δH 4.15 (br s), δC 73.0; δH 4.59 (br d, J = 2.4 Hz), δC
75.4] and an oxygenated quaternary carbon (δ 75.2) reso-
nances. In the 1H–1H COSY spectrum, the methylene protons at
δ 2.56 (1H, br d, J = 13.8 Hz) and 2.05 (1H, br d, J = 13.8 Hz)
were shown to be coupled to both of the two oxymethine
protons at δ 4.15 and δ 4.59. The oxymethine proton at δ 4.59
was further coupled to the methylene protons at δ 2.34 (1H, dd,
J = 13.8, 2.4 Hz) and 2.19 (1H, br d, J = 13.8 Hz). In the HMBC
spectrum, the long-range correlations from the oxymethine
proton at δ 4.15 to the carbons at δ 75.4 (CH), 75.2 (C), 44.0 (C),
33.2 (CH2), and 13.6 (CH3), from the oxymethine proton at δ
4.59 to the carbons at δ 73.0 (CH) and 75.2 (C), and from the
exchangeable proton at δ 6.56 (br s) to the carbons at δ 75.2 (C),
44.0 (C), and 35.4 (CH2) were observed. These findings allowed
the placement of these three hydroxyl groups at C-1, C-3 and C-
5 positions. HMBC correlation of the anomeric proton signal at
comparison of the 1H and 13C NMR chemical shifts with
literature data [3]. The HMBC correlation of the anomeric
proton signal at δ 4.91 with δ 158.5 (C-4′) proved the location of
the glucopyranosyl moiety at C-4′ of aglycone (Fig. 2). There-
fore, by combination of 1H–1H COSY, HSQC, HMBC and NOESY
spectra, the structure of compound 4 was determined to be
(+)-(2R,3R)-3,4′-dihydroxy-7-methoxy-8-methylflavan-4′-O-
β-D-glucopyranoside (tupichiside A).
All compounds (1–14) obtained were evaluated for their
in vitro growth inhibitory effects against Human LoVo and
BGC-823 cell lines using the MTT method as reported
previously, and six of them were found to possess potent
cytotoxicity. The cytotoxicity of the compounds against the
LoVo and BGC-823 cell lines was compared with the respective
positive control group Cisplatin, and finally, the result was
summarized in Table 3. The new compound 2 exhibited potent
toxicity effects against LoVo and BGC-823 cell lines, with IC50
values of 0.267 and 0.327 μM, respectively. Meanwhile,
compounds 8–9 also showed significantly cytotoxicity against
the selected cells, and the IC50 values were much lower than
that of Cisplatin on LoVo and BGC-823 cell lines, respectively.
Comparison of the cytotoxic activities and structures of
compounds 1–9 suggested that the ethylenic linkage at C-5
position increased their cytotoxic activities. In addition,
comparing compound
4 with 11, the flavane aglycone
δ
5.00 with
δ
75.4 (C-3) proved the location of the
tupichinol A (compound 11) possesses more cytotoxic activity
glucopyranosyl moiety at C-3 of aglycone (Fig. 2). The β-axial
orientations of all these hydroxyl groups were indicated by the
NOESY spectrum in which the NOE correlations were observed
between H-1/H-19, H-1/H2-2, H-3/H2-2, and H-3/H2-4, and
between the proton of the hydroxyl group at C-5 and H-4 eq
and H-19. On the basis of the above evidence, compound 3 was
determined to be 1β,3β,5β-triol-spirost-25(27)-ene-3-O-β-D-
glucopyranoside (tupistroside I).
than its glucoside.
Conflict of interest
We declare that we have no conflict of interest.
Acknowledgments
Compound 4 was obtained as yellow amorphous powder.
The HR-ESI-MS showed a [M + Na]+ ion at m/z 471.1629 (calcd
471.1626), consistent with the molecular formula C23H28O9. In
the 1H NMR spectrum of 4, signals that are characteristic of the
8-methylflavan-3-ol aglycone skeleton were observed [23].
Two signals at δ 2.10 (3H, s) and 3.78 (3H, s) were assigned to
the methyl group on C-8 and the methoxyl group attached to C-
7 in ring A, respectively. The oxymethine protons at δ 5.02 (1H,
s) and 4.22 (1H, br s) were assigned to H-2 and H-3,
respectively. The protons at δ 6.87 (1H, d, J = 8.4 Hz) and
6.52 (1H, d, J = 8.4 Hz) were assigned to H-5 and H-6,
respectively. Furthermore, the aromatic protons at δ 7.45 (2H,
d, J = 8.7 Hz) and 7.11 (2H, d, J = 8.7 Hz) were assigned to H-
2′/6′ and H-3′/5′, respectively [23]. The 13C NMR spectrum
showed the characteristic flavan-3-ol aglycone signals at δ 79.8,
67.5, and 34.4, corresponding to C-2 (OCH), C-3 (OCH), and C-4
(CH2), respectively [24,25]. Moreover, this spectrum also
indicated the required 12 aromatic carbons (δ 104.7-158.5),
one methoxyl carbon at δ 56.6, and one methyl carbon at δ 9.2.
In addition, the presence of a β-D-glucopyranosyl moiety in 4
was readily recognized by the appearance of an anomeric
proton signal at δ 4.91 (1H, d, J = 7.8 Hz) in the 1H NMR
spectrum and also by the characteristic six signals at δ 102.5
(CH), 75.0 (CH), 78.2 (CH), 71.5 (CH), 78.0 (CH) and 62.6 (CH2)
in the 13C NMR spectrum (Table 2). Acid hydrolysis of 4 with
2 N HCl gave D-glucose which was confirmed on TCL and
tupichinol A (compound 11) which was confirmed by direct
We are grateful to Mrs. Yan Xue, Mrs. Mei-feng Xu and
Mrs. Yu-mei Zhao of the National Center of Biomedical Analysis
for the measurements of the MS and NMR spectra.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.fitote.2015.02.008.
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