Four new furostanol saponins from the rhizomes and roots of Smilax scobinicaulis and their cytotoxicity

Article abstract of DOI:10.3390/molecules191220975

Four new furostanol saponins 1-4, along with two known furostanol saponins 5 and 6 and one known spirostanol saponin 7 were isolated from the rhizomes and roots of Smilax scobinicaulis. The structures of the new saponins were elucidated as 26-O-β-D-glucop

Full text of DOI:10.3390/molecules191220975

Molecules 2014, 19, 20975-20987; doi:10.3390/molecules191220975
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Four New Furostanol Saponins from the Rhizomes and Roots of
Smilax scobinicaulis and Their Cytotoxicity
Jing Xu 1,3, Shixiu Feng 2, Qi Wang 1, Yingli Cao 1, Miao Sun 1 and Cunli Zhang 1,*
1
College of Life Science, Northwest A&F University, Yangling 712100, Shaanxi, China;
E-Mails: seaqisky@163.com (J.X.); wangqidream2006@163.com (Q.W.);
caoyinglisky@163.com (Y.C.); sunmiao151@163.com (M.S.)
2
Key Laboratory of Southern Subtropical Plant Diversity, Shenzhen Fairy Lake Botanical Garden,
Chinese Academy of Sciences, 160 Xianhu Road, Liantang, Shenzhen 518004, China;
E-Mail: shixiufeng7787@gmail.com
3
Shenzhen Boton Flavors & Fragrances Co., Ltd., 19 Daxin Road, Nanshan,
Shenzhen 518051, China
* Author to whom correspondence should be addressed; E-Mail: cunli_zhang@nwsuaf.edu.cn;
Tel.: +86-137-7217-8964.
External Editor: Derek J. McPhee
Received: 15 September 2014; in revised form: 21 November 2014 / Accepted: 2 December 2014 /
Published: 15 December 2014
Abstract: Four new furostanol saponins 1–4, along with two known furostanol saponins
5 and 6 and one known spirostanol saponin 7 were isolated from the rhizomes and roots of
Smilax scobinicaulis. The structures of the new saponins were elucidated as
26-O-β-D-glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-22-methoxyl-6-one-3-O-α-
L-arabinopyranosyl-(1→6)-β-D-glucopyranoside (1), 26-O-β-D-glucopyranoside-3β,26-
dihydroxy-(25R)-5α-furostan-22-methoxyl-6-one (2), 26-O-β-D-glucopyranoside-3β,26-
dihydroxy-(25R)-5α-furostan-20(22)-en-6-one (3), 26-O-β-D-glucopyranoside-3β,23,26-
trihydroxy-(23R, 25R)-5α-furostan-20(22)-en-6-one (4) on the basis of spectroscopic
analysis. The isolated saponins were evaluated for cytotoxic activity against two human
cancer cell lines including Hela (cervical carcinoma) and SMMC-7221 (hepatocellular
carcinoma). Compounds 1 and 7 demonstrated cytotoxicity against the tested cell lines.
Keywords: Smilax scobinicaulis; furostanol saponins; cytotoxic activity

Molecules 2014, 19
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1. Introduction
The genus Smilax (family Liliaceae) is mainly distributed in the tropical, subtropical and temperate
areas of the world [1]. It is well known that steroidal saponins are abundant in the genus Smilax [2].
Many species of this genus have a long history of use as herbal remedies. Smilax scobinicaulis (C.H.)
Wright, commonly known as Hei Ci Ba Qia in Chinese is one of them, which is distributed in Shaanxi,
Gansu, Sichuan, Yunnan and other regions of China [3]. The rhizomes and roots of this plant, known
as “Jin Gang Teng” and “Wei Ling Xian” in North China, have long been used in folk medicine for the
treatment of rheumatic arthritis, lumbago, gout, tumor and inflammatory diseases [1]. Previous
phytochemical investigations of this plant led to the isolation of steroidal saponins [4], flavonoids [5]
and phenylpropanoids [6]. Bioactivity investigations showed that some of the isolated steroidal
saponins had antimicrobial and cytotoxic activity [4,7].
In a continuing search for new steroidal saponins from this plant, a series of steroidal saponins have
now been obtained, including four new furostanol saponins 1–4, two known furostanol saponins 5 and
6 and one known spirostanol saponin 7 (Figure 1), which are reported here as S. scobinicaulis
compounds for the first time. All the compounds have been tested for cytotoxicity against Hela and
SMMC-7221 human cancer cell lines. In this paper, we report the isolation and cytotoxic activity of
these compounds and the structural elucidation of the new compounds.
Figure 1. Chemical structure of compounds 1–7.
HO
HO
HO
HO
1"'
O
O
HO
HO
H
H
O
23
O
21
18
26
24
20OH
22
OOR2
25
27
H
OH
H
12
11
17
R2
19
13
O
H
H
1
9
16
H
14
2
H
15
10
8
7
H
O
H
5
3
H
O
H
R1O
R1O
4
6
H
H
1 R1=S R2=CH3
3 R1=H R2=H
R1=H R2=CH3
R1=H R2=OH
2
4
5 R1=S R2=H
6 R1=S R2=H
O
CH2OH
OH
O
H
O
1"
O
S=
HO
O
OH
H
H
HO
HO
1'
SO
OH
H
O
7
2. Results and Discussion
The rhizomes and roots of S. scobinicaulis were extracted with ethanol (EtOH). The extract was
suspended in water (H2O) and partitioned successively with petroleum ether (PE), ethyl acetate
(EtOAc) and n-butanol (n-BuOH). The n-BuOH soluble fraction was subjected to silica gel CC
(column chromatography), Sephadex LH-20, reversed phase silica gel CC and semi-preparative High
Performance Liquid Chromatography (HPLC) to afford four new furostanol saponins 26-O-β-D-

Molecules 2014, 19
20977
glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-22-methoxyl-6-one-3-O-α-L-arabinopyranosyl-
(1→6)-β-D-glucopyranoside (1), 26-O-β-D-glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-22-
methoxyl-6-one (2), 26-O-β-D-glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-20(22)-en-6-one
(3), 26-O-β-D-glucopyranoside-3β,23,26-trihydroxy-(23R, 25R)-5α-furostan-20(22)-en-6-one (4),
along with two known furostanol saponins 26-O-β-D-glucopyranosyl-3β,22ξ,26-trihydroxy-(25R)-5α-
furostan-6-one-3-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside (5) [8], 26-O-β-D-gluco-
pyranosyl-3β,26-dihydroxy-(25R)-5α-furostan-20(22)-en-6-one-3-O-α-L-arabinopyranosyl-(1→6)-β-D-
glucopyranoside (6) [9] and the known spirostanol saponin sieboldogenin-3-O-α-L-arabino-pyranosyl-
(1→6)-β-D-glucopyranoside (7) [10].
2.1. Structure Elucidation
Compound 1 was isolated as a white amorphous powder and showed a positive reaction (red colour)
to the Ehrlich reagent. The molecular formula, C45H74O19, was established from a positive molecular
ion peak at m/z 941.4727 [M+Na]+ (calcd. for C45H74NaO19: 941.4717) in the HR-ESI-MS spectrum
and supported by the ESI-MS (m/z 941.4 [M+Na]+ and m/z 917.3 [M−H]−) spectrum. Its IR spectrum
displayed strong absorption bands for hydroxyl groups at 3408 cm−1, for carbonyl group at 1707 cm−1
1
13
and absorption bands of alkyl groups at 2927 cm−1. The H- and C-NMR assignments of 1 were
based on the DEPT and 2D-NMR (COSY, HSQC, HMBC and NOESY) experiments and with the
positive red colour reaction in Ehrlich’s test suggested 1 was a furostanol saponin. To be specific, The
1H-NMR spectrum of 1 showed four methyl proton signals including two tertiary methyl groups at δH
0.86 (3H, s, H-18) and 0.80 (3H, s, H-19) and two secondary methyl groups at δH 1.04 (3H, d, J = 6.0 Hz,
H-21) and 0.98 (3H, d, J = 5.0 Hz, H-27), corresponding to C-atom signals at δC 15.5, 12.1, 14.7 and
15.9 in HSQC spectrum, typical steroid methyl signals. Besides, a signal for carbonyl at δC 212.0 was
present in the 13C-NMR. A correlation signal between H-7 at δH 2.16 and C-6 at δC 212.0 was observed
in the HMBC spectrum (Figure 2), which suggested the carbonyl is located at C-6. Moreover, a
1
methoxyl group at δH 3.17 (3H, s, OCH3) was also observed in the H-NMR spectrum. In the HMBC
spectrum, the correlations from OCH3 at δH 3.17 to C-22 at δC 112.6 to suggested the OCH3 was linked
to the C-22 (Figure 2). The configurations of 1 were mainly determined by NOESY spectrum. A
NOESY correlation signal between the H-5 proton at δH 2.40 and the H-9 proton at δH 1.40 was
consistent with the 5α configuration. A NOESY cross-peak between H-5 (δH 2.40) and H-3 (δH 3.36)
indicated that H-3 was α configuration. In addition, The NOE correlation between H-18 (δH 0.86, 3H, s)
and H-20 (δH 2.21, 1H, m) suggested that the C-21 methyl group was α-configuration. Thus, the
α-configurations of H-17 and the methoxy at C-22 position were unambiguously deduced based on the
strong NOE correlations of H-21/H-17 and H-21/OCH3. The 14α configuration was further confirmed
by the NOE correlations of H-14/H-16 and H-16/OCH3 [11]. However, the C-25 configuration of 1
was assigned as 25R based on the observed difference (∆ab = δa − δb = 0.35) of the 1H-NMR chemical
shifts of the H2-26 geminal protons, which was in agreement with that of 25R furostane-type steroidal
saponins (∆ab < 0.48 for 25R; ∆ab > 0.57 for 25S) [12].

Molecules 2014, 19
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Figure 2. Key HMBC correlations of compounds 1–4.
OH
OH
OH
OH
OH
OH
O
O
O
O
H
H
OH
OH
OCH3
OCH3
O
O
H
H
OH
O
O
H
H
H
O
H
OH
O
HO
O
OH
OH
H
H
O
OH
OH
2
1
OH
OH
OH
OH
OH
OH
O
O
O
O
H
H
OH
OH
OH
O
O
H
H
H
H
H
H
HO
HO
H
H
O
O
4
3
In addition, three anomeric proton signals at δH 4.42 (1H, d, J = 6.5 Hz, glc-1'), 4.34 (1H, d, J = 6.0 Hz,
1
ara-1'') and 4.26 (1H, d, J = 7.0 Hz, glc-1''') existed in the H-NMR spectrum and the HSQC showed
correlations with anomeric carbon signals at δC 100.9, 103.8 and 103.2, respectively, indicating the
1
13
presence of three sugar moieties. Combined the H-NMR, C-NMR (including DEPT) and 2D-NMR
(HSQC, HMBC and COSY), two glucopyranosyls and one arabinopyranosyl were found. The relative
configurations of the two glucopyranose moieties were all assigned as β-configurations based on their
coupling constants (J = 6.5 Hz, Glc-1'; J = 7.0 Hz, Glc-1''') of the anomeric protons. The relative
configuration of the arabinopyranose moiety was determined as α-confirguration by the coupling
constants (J = 6.0 Hz, Ara-1''). The sugars were determined to be D-glucose and L-arabinose by acid
hydrolysis of 1 and then trimethylsilylation and GC analysis on a chiral column in comparison with an
authentic sample. Furthermore, the location of the glucopyranosyl was found to be C-26 of the aglycon
on the basis of a glycosylation shift of C-26 at δC 74.6 and the HMBC correlation peak between H-1'''
of glucopyranosyl at δH 4.26 and C-26 of the aglycone at δC 74.6, which is a structural feature in plant
furostanol saponins [11]. The attachments of sugar chain were deduced from the HMBC spectrum, in
which the long range correlation between glc-H-1' at δH 4.42 with C-3 at δC 76.8 of the aglycone and
ara-H-1'' at δH 4.34 with C-6' at δC 68.2 of the glucopyranosyl were observed (Figure 2), respectively.
These results implied that the glucopyranosyl was linked to C-3 of the aglycone and the two sugars
were the 1→6 linkage. Thus, the structure of 1 was elucidated as 26-O-β-D-glucopyranoside-3β,26-
dihydroxy-(25R)-5α-furostan-22-methoxyl-6-one-3-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside.
Compound 2, obtained as a white amorphous powder, gave a positive red colour reaction in
Ehrlich’s test. The HR-ESI-MS spectrum of 2 showed a positive ion [M+Na]+ at m/z 647.3762,
consistent with the molecular formula C34H56O10 (calcd. for C34H56NaO10: 647.3766), which was
supported by the ESI-MS (m/z 647.4 [M+Na]+ and m/z 623.4 [M–H]−) spectrum. In the IR spectrum,

Molecules 2014, 19
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the glycosidic nature of 2 was shown by strong hydroxyl groups absorptions at 3423 cm−1, a carbonyl
group at 1713 cm−1 and the alkyl groups at 2927, 2872 and 2853 cm−1 was also displayed in the IR
spectrum. Compound 2 was suggested to be a furostanol glycoside with one sugar moiety by Ehrlich’s
1
13
test and the H- and C-NMR assignments of 2 based on the DEPT and 2D-NMR (COSY, HSQC,
HMBC and NOESY) experiments. The aglycone of 2 was deduced as the same as that of 1 by
1
comparison the H- and 13C-NMR spectra of 2 with those of 1. This was further confirmed by
comparison Rf values of the aglycones obtained by acid hydrolysis of 1 and 2 on TLC. The only one
sugar moiety was assigned to C-26 by a glycosylation shift of C-26 at δC 74.6 and the HMBC
correlation between H-1 of the glucopyranosyl at δH 4.26 and C-26 at δC 74.6 (Figure 2). The relative
configuration of the glucopyranose moiety was determined as β by the coupling constant (J = 8.0 Hz,
Glc-1''') of the anomeric proton. The glucopyranose moiety was determined as D-glucose by acid
hydrolysis of 2 and then trimethylsilylation and GC analysis on a chiral column in the same manner.
The 25R configuration of 2 was deduced by the difference (∆ab = 0.35) of the chemical shifts of the
H2-26 geminal protons. Thus, the structure of 2 was elucidated as 26-O-β-D-glucopyranoside-3β,26-
dihydroxy-(25R)-5α-furostan-22α-methoxyl-6-one.
Compound 3 was obtained as a white amorphous powder and positive ion HR-ESI-MS provided an
[M+Na]+ ion m/z 615.3510, corresponding to a molecular formula of C33H52O9 (calcd. for C33H52NaO9:
615.3504), which was supported by m/z 615.3 [M+H] + and m/z 591.3 [M–H]− in the ESI-MS spectrum.
The IR spectrum showed the presence of hydroxyl groups at 3421 cm−1, a carbonyl group at 1697 cm−1
and alkyl groups at 2943 cm−1, 2915 cm−1and 2865 cm−1. It was apparent from the NMR spectroscopic
13
data (1H, C, COSY, HSQC, HMBC and NOESY) of 3 that this compound differed from 2 only by
the presence of the ∆20,22 double bond. The occurrence of a ∆20,22 double bond was confirmed from the
HMBC spectrum which showed significant correlation peaks between the methyl proton signal of
CH3-21 with the carbon resonances of C-20 (δC 103.6) and C-22 (δC 151.8) (Figure 2). The sugar
moiety also was assigned as β-glucopyranose based on the coupling constant (J = 8.0 Hz, Glc-1''') of
the anomeric proton. Its absolute configuration was also a D- as determined by acid hydrolysis of 3 and
then trimethylsilylation and GC analysis on a chiral column. The α-configuration of H-17 was
determined by the NOE correlations of H-14/H-17. The 25R configuration of 3 was assigned by the
observed difference (∆ab = δa − δb = 0.32) of the 1H-NMR chemical shifts of the H2-26 geminal protons
(∆ab < 0.48 for 25R; ∆ab > 0.57 for 25S). Therefore, compound 3 was identified as 26-O-β-D-
glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-20(22)-en-6-one.
Compound 4, a white amorphous powder, was assigned as C33H52O10 on the basis of its HR-ESI-MS
data [M+Na]+ m/z 631.3450 (calcd. for C33H52NaO10: 631.3453) and its ESI-MS data m/z 631.55
[M+Na]+, 609.34 [M+H]− and 1215.03 [2M−H]−. The IR spectrum showed absorptions for hydroxyl
groups (3422 cm−1), carbonyl group (1708 cm−1) and alkyl groups (2934 cm−1 and 2870 cm−1). The 1D
and 2D NMR spectroscopic data of 4 were in good agreement with those of 3 except for noticeable
differences in the δH-23 and δC-23 of the aglycone. The sugar moiety also was determined as
β-D-glucopyranose by the coupling constant (J = 8.0 Hz, Glc-1''') of the anomeric proton and GC
analysis of hydrolysis followed trimethylsilylation of 4 in comparison with an authentic sample. The
configurations of H-17 and the C-25 of 4 was assigned as the same as those of 3 by the NOE
1
correlation of H-14/H-17 and the ∆ab = 0.24 of the two geminal protons of H2-26. In the H-NMR
spectrum, the proton signal at δH 4.91 (1H, t-like, J = 7.0 Hz, H-23), corresponded C-atom signal at δC

Molecules 2014, 19
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63.6 in the HSQC spectrum showed the presence of the hydroxyl at the C-23. On the basis of this
evidence, the structure of 4 was revised as 26-O-β-D-glucopyranoside-3β,23,26-trihydroxy-(25R)-5α-
furostan-20(22)-en-6-one. Besides the four new furostanol saponins, three known steroid saponins
were isolated from this plant. Their structures were identified by compared the NMR properties with
the reported data.
2.2. Cytotoxic Activity
The cytotoxicity of compounds 1–7 were tested against human cervical carcinoma cells (Hela) and
human hepatocellular carcinoma cells (SMMC-7221) by the MTT method. The results revealed that
compounds 2–6 were inactive (IC50 > 100 µM), while compounds 1 and 7 displayed cytotoxicity
against Hela carcinoma cell lines with IC50 values of 18.79 ± 1.12 μM and 9.73 ± 1.64 μM, respectively
and against SMMC-7221 cancer cell lines with IC50 values of 28.57 ± 1.57 μM and 21.54 ± 1.64 μM,
respectively. Cisplatin used as the positive control had the IC50 values of 5.12 ± 0.26 μM (Hela) and
14.23 ± 0.44 μM (SMMC-7221). This investigation indicated that compound 7 which displays a
spirostanol saponin structure showed stronger cytotoxic activity, whereas compound 1, with a
furostanol saponin structure, displayed moderate cytotoxic activity. Thus, according to our results, the
spirostanol saponin skeleton exhibits higher cytotoxic activity than furostanol saponins, which is
consistent with the literature [13].
3. Experimental Section
3.1. General
Optical rotations were measured with a Rudolph Research Analytical Autopol III automatic
polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA) with MeOH as solvent. The IR
spectra were conducted in KBr on a Bruker Tensor 27 spectrophotometer (Bruker, Billerica, MA, USA).
The UV spectra were recorded on a Thermo Scientific Evolution 300 UV-visible spectrophotometer
1
13
(Thermo Fisher Scientific, Waltham, MA, USA). The H- (500 MHz), C- (125 MHz) and 2D-NMR
spectra were recorded on a Bruker DRX-500 instrument using TMS as internal standard. ESI-MS were
collected on a Thermo Fisher LCQ Fleet mass spectrometer instrument (Thermo Fisher Scientific).
HR-ESI-MS were carried out on a Bruker Bio-TOF-IIIQ mass spectrometer (Bruker). Absorbance
value was measured by a Bio-Tek Epoch Absorbance Microplate Reader to calculate the inhibition rate
(Bio-Tek, Winooski, VT, USA).
Semi-preparative HPLC was run with a Waters 1525 pump and a Waters 2489 ultraviolet-visible
detector using an Xterra® prep MS C18 column (10 µm, 7.8 × 150 mm) (Waters, Milford, MA, USA).
For column chromatography, silica gel (200–300 mesh; Qingdao Haiyang Chemical Co. Ltd, Qingdao,
China), Rp-C18 (ODS-A, 50 µm, YMC, Yantai, China), and Sephadex LH-20 (GE Healthcare
Bio-Science AB, Fairfield, CT, USA) were used. TLC (5 × 10 cm plates) were performed on GF254
(Qingdao Haiyang Chemical Co. Ltd) plates. Spots were detected after spraying with Ehrlich reagent
(1 g 4-dimethylaminobenzaldehyde, in a mixture of 25 mL 37% hydrochloric acid and 75 mL
methanol) followed by heating.

Molecules 2014, 19
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3.2. Plant Material
The rhizomes and roots of S. scobinicaulis were collected from Taibai Mountain of Shaanxi
Province, China, in October 2010. The plant was identified by Zhenghai Wu of Northwest A&F
University, Yangling, Shannxi, China. An authenticated voucher specimen (XB 00045) has been
deposited at the Herbarium of Northwest A&F University, Yangling, Shannxi, China.
3.3. Extraction and Isolation
The air-dried powdered rhizomes and roots of S. scobinicaulis (62 kg) was extracted three times
with 70% EtOH at room temperature for 48 h to obtain a concentrated extract, which was suspended in
H2O and partitioned successively with PE, EtOAc and n-BuOH. The n-BuOH soluble extract (1792 g)
was subjected to D101 macroporous adsorption resin and successively eluted with 30% EtOH, 50%
EtOH and 70% EtOH to afford three fractions Frs.1–3. Fr.2 (600 g) was subjected to silica gel CC
using a stepwise gradient of EtOAc–MeOH–H2O (from 15:1:0.5 to 3:1:0.5) to afford ten sub-fractions
Frs. 2a–2j. Fr.2d (9.7 g) was chromatographed on silica gel CC eluting with EtOAc–MeOH (from 17:1
to 3:1) in gradient to yield four sub-fractions Frs.2d1–2d4 based on TLC. Fr.2d2 (1.2 g) was applied to
Sephadex LH-20 CC with the eluent of MeOH to yield four sub-fractions Frs.2d2a–2d2d. Fr.2d2a
(408 mg) was purified by RP-C18 CC and eluted with 65% MeOH–H2O to give compound 3 (52.7 mg).
Fr.2d3 (1.6 g) was applied to Sephadex LH-20 CC with the eluent of MeOH to yield three
sub-fractions Frs.2d3a–2d3c. Fr.2d3a (635 mg) was subjected to RP-C18 CC and eluted with 30%–70%
MeOH–H2O to result four sub-fractions Frs.2d3a1–2d3a4. Fr.2d3a2 (156 mg) was purified by
semi-preparative RP-C18 HPLC with the eluent of 48% MeOH–H2O (215 nm, flow rate: 3 mL/min) to
give compound 4 (43.6 mg, tR = 10.4 min). Fr.2d3a3 (238 mg) was purified by semi-preparative
RP-C18 HPLC with the eluent 55% MeOH-H2O (215 nm, flow rate: 5 mL/min) to give compound 2
(55.8 mg, tR = 7.9 min). Fr.2i (24.0 g) was chromatographed on Sephadex LH-20 CC with the eluent of
MeOH to yield five sub-fractions Frs.2i1–2i5 based on TLC. Fr.2i1 (2.1 g) was applied to RP-C18 CC
and eluted with 30%–70% MeOH–H2O to yield four sub-fractions Frs.2i1a–2i1d. Fr.2i1c (1.1 g) was
chromatographed on silica gel CC eluting with EtOAc–MeOH–H2O (7:1:0.3–2:1:0.3) in gradient to
yield four sub-fractions Frs.2i1c1–2i1c4. Fr.2i1c3 (223 mg) was purified by semi-preparative RP-C18
HPLC with the eluent of 53% MeOH–H2O (215 nm, flow rate: 3 mL/min) to give compound 5
(41.7 mg, tR = 8.7 min) and 1 (42.3 mg, tR = 9.3 min). Fr.2i1d (0.8 g) was chromatographed on silica
gel CC eluting with EtOAc–MeOH–H2O (from 15:2:0.3 to 7:2:0.3) in gradient to give compound 7
(43.6 mg) and to yield five sub-fractions Frs.2i1d1-2i1d4. Fr.2i1d3 (339 mg) was purified by
semi-preparative RP-C18 HPLC with the eluent of 50% MeOH–H2O (215 nm, flow rate: 2.5 mL/min)
to give compound 6 (75.2 mg, tR = 8.3 min).
3.4. New Compound Data
26-O-β-D-Glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-22-methoxyl-6-one-3-O-α-L-arabino-
23.4
pyranosyl-(1→6)-β-D-glucopyranoside (1). White amorphous powder, mp > 290 °C; [α]D = −53.348°
1
13
(c 0.115, CH3OH); IR υmax (KBr): 3408, 2927, 1707 and 1044 cm−1; H-NMR and C-NMR data see

Molecules 2014, 19
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Table 1; ESI-MS: m/z 941.4 [M+Na]+ and 917.3 [M−H]−; HR-ESI-MS: m/z 941.4727 [M+Na]+ (Calcd
for C45H74NaO19: 941.4717).
Table 1. 1H- and 13C-NMR Data of Compounds 1–4. (δ in ppm, J in Hz).
1
2
3
4
NO.
1
a
b
a
b
a
b
c
d
δH
δC
δH
δC
δH
δC
δH
δC
1.80 (m)
1.35 (m)
1.93 (m)
1.32 (m)
3.36 (m)
1.43 (m)
2.03 (m)
1.79 (m)
1.33 (m)
1.82 (m)
1.64 (m)
3.53 (m)
1.41 (m)
1.81 (m)
2.38 (d, 11.0)
–
1.80 (m)
1.32 (m)
1.42 (m)
1.31 (m)
3.52 (m)
1.43 (m)
1.83 (m)
1.64 (m)
1.16 (m)
1.65 (m)
2.04 (m)
3.83 (m)
1.90 (m)
2.29 (m)
2.26 (m)
–
36.3
36.3
36.3
36.8
2
3
4
28.6
76.8
26.0
30.1
69.7
29.3
30.1
69.7
29.3
31.6
69.8
31.0
5
6
2.40 (d, 12.0) 56.0
56.2 2.37 (dd, 12.5, 2.0) 56.2
56.8
–
212.0
212.0
–
2.15 (m)
2.28 (dd, 13.0, 4.5)
1.97 (m)
1.36 (m)
–
212.0
209.7
2.16 (t, 13.0)
2.24 (m)
2.00 (m)
1.40 (m)
–
2.14 (t, 12.5)
2.25 (dd, 13.0, 4.5)
2.00 (m)
1.40 (m)
–
2.02 (m)
2.35 (m)
1.81 (m)
1.15 (m)
–
7
46.1
46.1
46.2
46.8
8
9
37.4
53.4
40.8
37.4
53.4
40.6
37.3
53.4
40.6
37.0
53.5
40.7
10
1.42 (m)
1.70 (m)
1.28 (m)
1.82 (m)
–
1.42 (m)
1.68 (m)
1.25 (m)
1.82 (m)
–
1.42 m
1.72 m
1.37 m
1.88 m
–
1.52 (m)
1.73 (m)
11
12
21.0
39.1
21.0
39.2
21.2
39.0
21.5
39.2
1.22 (m)
13
14
41.1
56.0
41.1
56.0
43.6
54.6
–
43.9
54.6
1.43 (m)
1.30 (m)
1.95 (m)
4.40 (m)
1.80 (m)
0.86 (s)
0.80 (s)
2.21 (m)
1.04 (d, 6.0)
–
1.41 (m)
1.30 (m)
1.94 (m)
4.41 (q-like, 7.5)
1.79 (m)
0.85 (s)
1.45 (m)
1.45 (m)
2.15 (m)
4.78 (m)
2.56 (d, 10.0)
0.72 (s)
0.79 (s)
–
1.06 (m)
1.38 (m)
1.97 (m)
4.78 (m)
15
31.1
31.2
33.5
84.0
33.9
84.1
16
17
18
19
20
21
22
80.8
63.6
15.5
12.1
39.8
14.7
112.6
80.6
63.6
15.5
12.1
39.8
14.5
112.6
64.0 2.48 (d, 10.5) 64.5
13.3
12.0
0.66 (s)
0.75 (s)
–
14.2
13.0
0.79 (s)
2.21 (m)
1.03 (d, 7.0)
–
103.6
10.5
104.6
11.4
1.63 (s)
–
1.76 (s)
151.8
154.3
1.63 (m)
1.84 (m)
1.16 (m)
1.62 (m)
1.76 (m)
3.40 (m)
3.76 (m)
0.98 (d, 5.0)
3.17 (s)
1.62 (m)
1.81 (m)
1.16 (m)
1.61 (m)
1.75 (m)
3.40 (dd, 9.5, 6.5)
3.75 (dd, 9.5, 6.5)
0.97 (d, 7.0)
3.17 (s)
23
29.9
29.9
2.16 (m)
22.7
4.91 (t, 7.0)
63.6
1.27 (m)
1.65 (m)
1.74 (m)
2.38 (m)
2.44 (m)
3.79 (m)
4.03 (m)
1.17 (d, 6.0)
–
24
25
26
27.6
33.6
74.6
27.2
33.6
74.6
30.6
32.7
74.4
39.5
30.7
75.3
1.78 (m)
3.41 (dd, 9.5, 6.0)
3.73 (dd, 9.5, 7.0)
0.97 (d, 6.5)
–
27
15.9
46.3
16.0
46.2
–
15.9
–
17.6
–
OCH3
Glc-1'
4.42 (d, 6.5) 100.9
–
–
–
–
–

Molecules 2014, 19
20983
Table 1. Cont.
1
2
3
4
NO.
a
b
a
b
a
b
c
d
δH
δC
δH
–
δC
δH
–
δC
–
δH
–
δC
2'
3'
4'
5'
3.19 (m)
73.7
76.6
70.4
75.5
–
–
–
–
–
–
–
–
3.37 (m)
3.30 (m)
–
–
–
–
–
–
–
–
3.45 (m)
–
–
–
–
3.83 (m)
6'
68.2
–
–
–
–
–
–
4.09 (d, 11.5)
Ara-1''
2''
4.34 (d, 6.0) 103.8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3.60 (m)
3.54 (m)
3.71 (m)
3.88 (m)
71.0
72.8
68.0
65.2
3''
4''
5''
Glc-1''' 4.26 (d, 7.0) 103.2
4.26 (d, 8.0)
3.20 (t, 8.0)
103.2
73.8
76.5
70.3
76.7
4.25 (d, 8.0)
3.21 (t, 8.5)
103.1 4.85 (d, 8.0) 104.8
2'''
3'''
4'''
5'''
3.37 (m)
3.73 (m)
3.30 (m)
3.29 (m)
3.67 (m)
3.90 (m)
73.8
77.1
70.4
76.5
73.7
76.5
70.3
76.7
4.03 (m)
4.22 (m)
75.0
78.4
71.6
78.3
3.28 (m)
3.27 (m)
3.29 (m)
3.32 (m)
4.19 (m)
3.35 (m)
3.37 (m)
3.94 (m)
3.68 (dd, 12.0, 5.5)
3.88 (dd, 12.0, 1.5)
3.70 (dd, 12.0, 6.0)
3.88 (dd, 12.0, 2.0)
4.36 (m)
6'''
61.5
61.4
61.4
62.7
4.54 (d, 11.0)
a: Recorded at 500 MHz in CD3OD; b: Recorded at 125 MHz in CD3OD; c: Recorded at 500 MHz in Pyridine-d5;
d: Recorded at 100 MHz in Pyridine-d5.
26-O-β-D-Glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-22-methoxyl-6-one (2). White amorphous
23.8
powder, mp > 290 °C; [α]D = −40.029° (c 0.119, CH3OH); IR υmax (KBr): 3423, 2927, 2872, 2853,
1713 and 1042 cm−1; 1H-NMR and 13C-NMR data see Table 1; ESI-MS: m/z 647.4 [M+Na]+ and 623.4
[M−H]−; HR-ESI-MS: m/z 647.3762 [M+Na]+ (Calcd for C34H56NaO10: 647.3766).
26-O-β-D-Glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-20(22)-en-6-one (3). White amorphous
24.1
powder, mp > 290 °C; [α]D = −6.152° (c 0.115, CH3OH); IR υmax (KBr): 3421, 2943, 2915, 2865,
1
13
1697 and 1071 cm−1; H-NMR and C-NMR data see Table 1; ESI-MS: m/z 615.3 [M+Na]+, 591.3
[M−H]− and 1183.5 [2M−H]−; HR-ESI-MS: m/z 615.3510 [M+Na]+ (Calcd for C33H52NaO9: 615.3504).
26-O-β-D-Glucopyranoside-3β,23,26-trihydroxy-(23R,25R)-5α-furostan-20(22)-en-6-one (4). White
amorphous powder, mp > 290 °C; [α]20.8 D = −18.485° (c 0.13, CH3OH); IR υmax (KBr) (cm−1): 3422,
1
13
2934, 2870, 1708, and 1077 cm−1; H-NMR and C-NMR data see Table 1; ESI-MS: m/z 631.55
[M+Na]+, 609.34 [M+H]+ and 1215.03 [2M−H]−; HR-ESI-MS: m/z 631.3450 [M+Na]+ (Calcd for
C33H52NaO10: 631.3453).
26-O-β-D-Glucopyranosyl-3β,22ξ,26-trihydroxy-(25R)-5α-furostan-6-one-3-O-α-L-arabinopyranosyl-
(1→6)-β-D-glucopyranoside (5). A white amorphous powder, molecular formula was C44H72O19.
ESI-MS m/z 903.3 [M−1]−; 1H-NMR (CD3OD, 500 MHz) δ: 2.41 (1H, d, J = 12.0 Hz, H-5), 1.40 (1H,
m, H-9), 1.45 (1H, m, H-14), 4.61 (1H, d, J = 7.5, H-16), 1.83 (1H, m, H-17), 0.85 (3H, s, H-18), 0.80
(3H, s, H-19), 1.04 (3H, d, J = 6.5 Hz, H-21), 3.41 (1H, m, H-26a), 3.75 (1H, m, H-26b), 0.97 (3H, d,

Molecules 2014, 19
20984
J = 6.0 Hz, H-27), 4.42 (1H, d, J = 7.5 Hz, glc-H-1'), 4.10 (1H, d, J = 11.0 Hz, glc-H-6a'), 3.83 (1H, m,
glc-H-6b'), 4.35 (1H, d, J = 6.5 Hz, glc-H-1''), 4.26 (1H, d, J = 7.5 Hz, glc-H-1'''); 13C-NMR (CD3OD,
125 MHz) δ: 36.3 (C-1), 28.6 (C-2), 76.7 (C-3), 26.0 (C-4), 56.0 (C-5), 212.0 (C-6), 46.1 (C-7), 37.4
(C-8), 53.4 (C-9), 40.8 (C-10), 21.0 (C-11), 39.2 (C-12), 41.1 (C-13), 56.0 (C-14), 31.2 (C-15), 80.6
(C-16), 62.6 (C-17), 15.5 (C-18), 12.1 (C-19), 39.4 (C-20), 14.4 (C-21), 110.5 (C-22), 35.6 (C-23),
27.2 (C-24), 33.5 (C-25), 74.6 (C-26), 16.0 (C-27), 100.9 (glc-C-1'), 73.7 (glc-C-2'), 76.6 (glc-C-3'),
70.4 (glc-C-4'), 75.5 (glc-C-5'), 68.2 (glc-C-6'), 103.8 (ara-C-1''), 71.0 (ara-C-2''), 72.8 (ara-C-3''), 68.0
(ara-C-4''), 65.2 (ara-C-5''), 103.2 (glc-C-1'''), 73.8 (glc-C-2'''), 77.1 (glc-C-3'''), 70.3 (glc-C-4'''), 76.5
(glc-C-5'''), 61.4 (glc-C-6''').
26-O-β-D-Glucopyranosyl-3β,26-dihydroxy-(25R)-5α-furostan-20(22)-en-6-one-3-O-α-L-
arabinopyranosyl-(1→6)-β-D-glucopyranoside (6). A white amorphous powder, molecular formula
was C44H70O18. ESI-MS m/z 885.3 [M−1]−; 1H-NMR (CD3OD, 500 MHz) δ: 2.41 (1H, d, J = 11.0 Hz,
H-5), 2.27 (1H, dd, J = 13.0 and 4.5 Hz, H-7a), 2.19 (1H, m, H-7b), 1.41 (1H, m, H-9), 1.35 (1H, m,
H-14), 4.77 (1H, m, H-16), 2.56 (1H, d, J = 10.0, H-17), 0.73 (3H, s, H-18), 0.80 (3H, s, H-19), 1.64
(3H, s, H-21), 3.38 (1H, m, H-26a), 3.72 (1H, m, H-26b), 0.98 (3H, d, J = 6.5 Hz, H-27), 4.43 (1H, d,
J = 8.0 Hz, glc-H-1'), 4.10 (1H, d, J = 10.5 Hz, glc-H-6a'), 3.84 (1H, m, glc-H-6b'), 4.35 (1H, d,
J = 6.5 Hz, glc-H-1''), 4.26 (1H, d, J = 8.0 Hz, glc-H-1'''); 13C-NMR (CD3OD, 125 MHz) δ: 36.3 (C-1),
28.6 (C-2), 76.7 (C-3), 26.0 (C-4), 56.0 (C-5), 212.0 (C-6), 46.3 (C-7), 37.3 (C-8), 53.4 (C-9), 40.8 (C-10),
21.2 (C-11), 39.0 (C-12), 43.7 (C-13), 54.6 (C-14), 33.5 (C-15), 84.0 (C-16), 64.0 (C-17), 13.3 (C-18),
12.1 (C-19), 103.6 (C-20), 10.5 (C-21), 151.8 (C-22), 22.7 (C-23), 30.6 (C-24), 32.7 (C-25), 74.4 (C-26),
16.0 (C-27), 100.9 (glc-C-1'), 73.7 (glc-C-2'), 76.5 (glc-C-3'), 70.3 (glc-C-4'), 75.4 (glc-C-5'), 68.2 (glc-C-6'),
103.8 (ara-C-1''), 71.0 (ara-C-2''), 72.8 (ara-C-3''), 68.1 (ara-C-4''), 65.3 (ara-C-5''), 103.1 (glc-C-1'''),
73.7 (glc-C-2'''), 77.1 (glc-C-3'''), 70.1 (glc-C-4'''), 76.5 (glc-C-5'''), 61.4 (glc-C-6''').
Sieboldogenin-3-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside (7). A white amorphous powder,
1
molecular formula was C38H60O14. H-NMR (DMSO, 500 MHz) δ: 2.33 (1H, d, J = 12.0 Hz, H-5),
1.30 (1H, m, H-9), 1.32 (1H, m, H-14), 4.31 (1H, q-like, J = 7.5, H-16), 0.73 (3H, s, H-18), 0.67 (3H,
s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 3.41 (1H, m, H-26a), 3.51 (1H, m, H-26b), 3.73 (1H, d,
J = 10.0 Hz, H-27a), 3.49 (1H, m, H-27b), 4.23 (1H, d, J = 7.5 Hz, glc-H-1'), 3.89 (1H, d, J = 11.0 Hz,
glc-H-6a'), 3.49 (1H, m, glc-H-6b'), 4.21 (1H, d, J = 5.0 Hz, glc-H-1''); 13C-NMR (DMSO, 125 MHz)
δ: 36.5 (C-1), 29.1 (C-2), 76.1 (C-3), 26.6 (C-4), 55.7 (C-5), 210.4 (C-6), 46.4 (C-7), 37.2 (C-8), 53.0
(C-9), 40.8 (C-10), 21.0 (C-11), 39.4 (C-12), 40.9 (C-13), 56.0 (C-14), 31.6 (C-15), 80.6 (C-16), 62.1
(C-17), 16.6 (C-18), 13.3 (C-19), 42.0 (C-20), 15.0 (C-21), 109.4 (C-22), 26.7 (C-23), 21.3 (C-24),
35.2 (C-25), 60.5 (C-26), 60.3 (C-27), 100.9 (glc-C-1'), 73.9 (glc-C-2'), 77.1 (glc-C-3'), 70.7 (glc-C-4'),
75.9 (glc-C-5'), 68.7 (glc-C-6'), 103.9 (ara-C-1''), 71.0 (ara-C-2''), 73.0 (ara-C-3''), 67.7 (ara-C-4''),
65.2 (ara-C-5'').
3.5. Acid Hydrolysis and Sugar Analysis
Compounds 1–4 (30 mg each) in 1 M HCl (MeOH–H2O, 1:1) were heated under reflux for 8 h.
After removal of the solvent, the residue was partitioned between CHCl3 and H2O. The water layer was
neutralized with 5% NaOH and desalted (Sephadex LH-20, MeOH). The desalted segment was found

Molecules 2014, 19
20985
to contain D-glucose and L-arabinose by TCL comparison with authentic sample (EtOAc–MeOH–
H2O–AcOH = 6.5:2.0:1.5:1.5) and by measurement of their optical rotation ([α]20 D + 60°, c = 0.15,
20
H2O; [α]D = +118°, c = 0.15, H2O) for compound 1, and was only found to contain D-glucose for
compounds 2–4.
3.6. GC Analysis of the Sugar Moieties in 1–4
Compounds 1–4 (each 4 mg) in 1 M HCl (5 mL, dioxane–H2O 1:1, v/v) were heated at 95 °C for
6 h, respectively. The reaction mixtures were evaporated in vacuo. The residues were dissolved in
water and then extracted with CHCl3 for three times, respectively. After evaporating the aqueous phases
to dryness in vacuo, the residues were dissolved in pyridine (5 mL) and 1-(trimethylsilyl)-imidazole
(0.5 mL) at room temperature for 30 min. The reaction mixtures were dried with a stream of N2. The
residues were partitioned between CHCl3 and H2O. The organic layers were subjected to GC analysis
using an L-Chirasil-Val column (0.32 mm × 25 m) [14]. Temperature of the injector and detector was
200 °C. A temperature gradient system was used for the oven; the initial was temperature was
maintained at 100 °C for 1 min and then increasing up to 180 °C at a rate of 5 °C/min. Peaks of the
hydrolysate of 1 were detected at 8.92 and 9.80 min (L-arabinose) and 14.71 min (D-glucose).
However, respectively detecting only one peak at 14.73, 14.72 and 14.72 min in the hydrolysate of 2, 3
and 4, suggested that all the sugar moieties of 2, 3 and 4 are D-glucose. Retention times for authentic
samples of D-glucose and L-arabinose (Sigma Aldrich, St. Louis, MO, USA) after being treated in
the same manner with 1-(trimethylsilyl)-imidazole in pyridine were detected at 8.90 and 9.78 min
(L-arabinose), 14.71 min (D-glucose).
3.7. Cytotoxicity Assay
The cytotoxic activities of compounds 1–7 were performed against human cervical carcinoma cells
(Hela) and human hepatocellular carcinoma cells (SMMC-7221) by MTT [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide] assay [15], with cisplatin as positive control. Cell lines were
maintained in Dulbecco’s Modified Eagle Medium (DMEM) medium containing 10% fetal bovine
serum and were cultivated in humidified incubator at 5% CO2 and 37 °C. The human cancer cells in
the log phase of their growth cycle (2.5 × 104 cells/mL) were added to each well of the 96-well plates
(100 µL/well) and were cultivated for 16 h. The test compounds were then added and the cells were
further incubated for 48 h. After, 20 µL of MTT solution (5 mg/mL in PBS) were added to each well
and the cells were incubated for addtitional 4 h. The supernatant was discarded and after washing with
PBS, DMSO (100 µL/well) was added to dissolve the formazan crystals. The optical density (OD) was
measured by enzyme immunoassay instrument at 570 nm. Each assay was done in triplicate.
4. Conclusions
Four new furostanol saponins 1–4 were isolated and identified from the rhizomes and roots of
Smilax scobinicaulis. Besides, two known furostanol saponins 5–6 and one known spirostanol saponin
7 are described for the first time in this plant. Among the isolated saponins, compounds 1 and 7
displayed cytotoxicity against the Hela and SMMC-7221 cancer cell lines. The discovery of these new

Molecules 2014, 19
20986
bioactive compounds further expands our knowledge of the structural diversity of the bioactive
products produced by the plant Smilax scobinicaulis, and lays a chemical foundation for its
pharmacological application.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/12/20975/s1.
Acknowledgments
This research was supported by the National Natural Science Foundation of China (31070540).
Author Contributions
In this paper, the extraction and isolation were accomplished by Jing Xu, Qi Wang, Yingli Cao and
Miao Sun; Shixiu Feng and Jing Xu carried out the structural elucidations; Jing Xu also participated in
the MTT assay and analyzed the results; Jing Xu was in charge of writing the manuscript and Shixiu Feng
help revising the manuscript; As corresponding author Cunli Zhang organized the study.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–7 are available from the authors.
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