A new cytotoxic steroidal saponin from the rhizomes and roots of Smilax scobinicaulis

Article abstract of DOI:10.1080/14786419.2012.725396

A phytochemical investigation of the EtOH extract from the rhizomes and roots of Smilax scobinicaulis resulted in the isolation of a new isospirostanol-type steroidal saponin, namely (25 R)-5α-spirostan-3β, 6β-diol 3-O-β-D-glucopyranosyl-(1 → 4)-[α-L- arabinopyranosyl-(1 → 6)]-β-D-glucopyranoside (1), along with four known steroidal saponins (2-5). The structures of these compounds were determined by 1D- and 2D-NMR spectroscopic analysis, FABMS and HR-ESI-MS as well as chemical degradation. The isolated saponins were evaluated for their in vitro cytotoxicity against A549, LAC and Hela human cancer cell lines, which demonstrated that only compound 1 possessed significant cytotoxic activity with IC₅₀ values of 3.70, 5.70 and 3.64 μM, respectively.

Full text of DOI:10.1080/14786419.2012.725396

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A new cytotoxic steroidal saponin
from the rhizomes and roots of Smilax
scobinicaulis
a
b
a
c
Cunli Zhang , Shixiu Feng , Lanxi Zhang & Zhanjun Ren
a
College of Life Science , Northwest A&F University , Yangling ,
Shaanxi 712100 , China
b
Laboratory of Southern Subtropical Plant Diversity , Shenzhen
Fairy Lake Botanical Garden, Chinese Academy of Sciences , 160
Xianhu Road, Liantang, Shenzhen, 518004 , China
c
College of Animal Science and Technology , Northwest A&F
University , Yangling , Shaanxi 712100 , China
Published online: 17 Sep 2012.
To cite this article: Cunli Zhang , Shixiu Feng , Lanxi Zhang & Zhanjun Ren (2013) A new cytotoxic
steroidal saponin from the rhizomes and roots of Smilax scobinicaulis , Natural Product Research:
Formerly Natural Product Letters, 27:14, 1255-1260, DOI: 10.1080/14786419.2012.725396
To link to this article: http://dx.doi.org/10.1080/14786419.2012.725396
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Natural Product Research, 2013
Vol. 27, No. 14, 1255–1260, http://dx.doi.org/10.1080/14786419.2012.725396
A new cytotoxic steroidal saponin from the rhizomes and roots
of Smilax scobinicaulis
a
b
a
c
Cunli Zhang , Shixiu Feng , Lanxi Zhang and Zhanjun Ren *
a
College of Life Science, Northwest A&F University, Yangling, Shaanxi 712100, China;
Laboratory of Southern Subtropical Plant Diversity, Shenzhen Fairy Lake Botanical Garden,
b
Chinese Academy of Sciences, 160 Xianhu Road, Liantang, Shenzhen, 518004, China;
College of Animal Science and Technology, Northwest A&F University,
Yangling, Shaanxi 712100, China
c
(
Received 19 January 2012; final version received 5 August 2012)
A phytochemical investigation of the EtOH extract from the rhizomes and roots
of Smilax scobinicaulis resulted in the isolation of a new isospirostanol-type
steroidal saponin, namely (25 R)-5�-spirostan-3ꢀ,6ꢀ-diol 3-O-ꢀ-D-glucopyrano-
syl-(1 ! 4)-[�-L-arabinopyranosyl-(1 ! 6)]-ꢀ-D-glucopyranoside (1), along with
four known steroidal saponins (2–5). The structures of these compounds were
determined by 1D- and 2D-NMR spectroscopic analysis, FABMS and HR–ESI–
MS as well as chemical degradation. The isolated saponins were evaluated for
their in vitro cytotoxicity against A549, LAC and Hela human cancer cell lines,
which demonstrated that only compound 1 possessed significant cytotoxic
activity with IC50 values of 3.70, 5.70 and 3.64 mM, respectively.
Keywords: Smilax scobinicaulis; steroidal saponins; cytotoxicity
1
. Introduction
The genus Smilax (family Liliaceae) comprises about 79 native species in China (Delectis
florae reipublicae popularis sinicae agendae academiae sinicae edita, 1978), many of which
have been used as herbal remedies for cardiac and cerebrovascular diseases for a long
history. Smilax scobinicaulis (C.H.) Wright, also termed as Hei Ci Ba Qia in Chinese, is
used in traditional medicine for the treatment of rheumatic arthritis, lumbago, gout and
inflammatory diseases. Previous phytochemical investigations of this plant led to the
isolation of a furostanol glycoside (Liu, Fu, Gao, & Qiu, 2002), seven steroidal saponins
(Zhang, Li, Gao, & Fu, 2003; Zhang, Zhu, Cheng, & Li, 2003; Zhang et al., 2012). In our
continuing research for new anti-cancer steroidal saponin, a new cytotoxic isospirostanol-
type steroidal saponins namely (25 R)-5�-spirostan-3ꢀ,6ꢀ-diol 3-O-ꢀ-D-glucopyranosyl-
(
7
1 ! 4)-[�-L-arabinopyranosyl-(1 ! 6)]-ꢀ-D-glucopyranoside (1) was isolated from the
0% EtOH extract of the rhizomes and roots of S. scobinicaulis, along with four known
compounds (2–5; Figure 1).
*
Corresponding author. Email: renzhanjun@nwsuaf.edu.cn
©
2013 Taylor & Francis

1
256
C. Zhang et al.
Figure 1. Chemical structure of compounds 1–5.
2
. Results and discussion
Compound 1 was obtained as a white amorphous powder and showed a positive reaction
in the Liebermann–Burchard test. Its molecular formula was deduced as C H O from
4
4
72 18
�
�
the quasi-molecular ion peak at m/z 887.4638 ([M � H] , D � 0.2 ppm) in the HR–ESI–
MS, as well as the negative ion peak at m/z 887 [M � H] (1 0 0) in the FABMS. The IR
�
1
spectrum showed strong absorptions for hydroxyl groups (3421 and 1049 cm ). In the
1
0
H-NMR and HSQC spectra, three anomeric protons at � 4.93 (d, J ¼ 7.6 Hz, H-1 ), 5.54
H
00
000
(
with three anomeric carbon signals at � 102.1 (C-1 ), 104.9 (C-1 ) and 105.6 (C-1 ),
d, J ¼ 7.8 Hz, H-1 ) and 5.11 (d, J ¼ 7.5 Hz, H-1 ) were observed, which were correlated
0
00
000
C
respectively. Acid hydrolysis of 1 resulted in the production of D-glucose and L-arabinose,
which were confirmed by the combination of co-TLC comparison and GC analysis
1
3
(
Section 3). Furthermore, the NMR and DEPT spectra exhibited 44 carbon signals, of
which the distinctive quaternary carbon signal at � 109.3 (C-22) led to the hypothesis that
C
1
was a spirostanol triglycosides (Zhang et al., 2012).
1
Comparison of the H- and C-NMR assignments of 1 with those of eruboside B
13
reported from Allium erubescens previously (Ahmad & Basha, 2007), suggested the
aglycone of 1 was (25 R)-5ꢀ-spirostan-3ꢁ,6ꢁ-diol. Notably, the presence of a hydroxyl
group at C-6 of the aglycone in 1 was disclosed by a significant downfield resonance at
�C 71.9 (C-6), as well as by the HMBC correlations of H-6 (� 4.04) with C-4 (� 32.8), C-8
H
C
(
� 30.7) and C-10 (� 35.9). The CH -27 was unambiguously attached at C-25 by the
C C 3

Natural Product Research
1257
Figure 2. Key HMBC correlation (arrows) of compound 1.
deduction of correlations form H-27 (� 0.67) to C-24 (� 28.8) and C-26 (� 66.9)
H
C
C
(
carbon signals at � 49.0 (C-5), 54.5 (C-9) and 12.4 (C-19) in the C-NMR spectrum,
Figure 1). The A/B and B/C trans-rings fusion were concluded by observation of the
1
3
C
suggesting the 5 -ꢀ configuration of H-5, respectively (Zhou & Chen, 2008). The
ꢁ
1
-configurations of 3-OH and 6-OH were further confirmed by comparison of its H- and
1
3
C-NMR data with steroidal saponins from Smilax officinalis which demonstrated the
same stereochemical configuration for the A and B rings, as well as the cross peaks
between H-5 (� 0.96)/H-3 (� 3.83), and between H-5/H-6 in the NOESY spectrum
H
H
(
Bernardo, Pinto, & Parente, 1996). The 25 R configuration of the aglycone was confirmed
by the two proton signals of H-26 at � 3.59 (brd, J ¼ 10.5 Hz, H-26ax) and 3.51 (dd,
H
1
J ¼ 9.5, 11.0 Hz, H-26eq) in the H-NMR spectrum (Debella, Haslinger, Kunert, Michl, &
1
3
Abebe, 1999). Moreover, comparison of the C-NMR spectra of the aglycone portion of 1
with the known saponin from S. officinalis (25 S-spirostan-3ꢁ,6ꢁ-diol; no. 181375-68-8)
showed the downfield shifts for the C-23 (þ5.5 ppm), C-24 (þ2.6 ppm), C-25 (þ2.8 ppm)
and C-26 (þ1.8 ppm) due to the effect by the 25 R configuration of an methyl group at
C-25 (Bernardo et al., 1996), which were consistent with the literature reported
2
5 R-spirostans (Acharya et al., 2010; Ahmad & Basha, 2007; Agrawal et al., 1995).
The attachment of the oligosaccharidic chain and the interglycosidic linkage was
established by the analysis of HMBC experimental data of 1 as well as comparison to the
previously isolated steroid saponins smilscobinoside A (Zhang et al., 2012), which
demonstrated that they had the same sugar moieties. Briefly, long range correlation from
0
00
H-1 of the internal glucopyranose (Glc I) to C-3 (� 77.7) of the aglycone, from H-1 of
C
0
000
glucopyranose (Glc II) to C-4 (� 81.1) of Glc-I, and from H-1 of the arabinopyranose to
C
0
C-6 (� 68.4) of Glc-I proved the connections (Figure 2). Thus, the structure of 1 was
C
elucidated as (25 R)-5ꢀ-spirostan-3ꢁ,6ꢁ-diol 3-O-ꢁ-D-glucopyranosyl-(1 ! 4)-[ꢀ-L-arabi-
nopyranosyl-(1 ! 6)]-ꢁ-D-glucopyranoside.
The four known saponins (25 S)-spirostan-5-en-3ꢁ,27-diol-3-O-[ꢀ-L-arabinopyranosyl-
(
3
(
2
1 ! 6)]-ꢁ-D-glucopyranoside (2) (Ju & Jia, 1993), (25 R)-5ꢀ-spirostan-3ꢁ-ol (tigogenin)
-O-ꢁ-D-glucopyrnosyl-(1 ! 4)-[ꢀ-L-arabinopyranosyl-(1 ! 6)]-ꢁ-D-glucopyranoside (3)
Kubo, Mimaki, Sashida, Nikaido, & Ohmoto, 1992), (25 S)-spirostan-5-en-3ꢁ,17ꢀ,
7-triol 3-O-[ꢀ-L-arabinopyranosyl-(1 ! 6)]-ꢁ-D-glucopyranoside (4) (Jia & Ju, 1992),
(
25 S)-spirostan-5-en-3ꢁ,17ꢀ,27-triol 3-O-ꢁ-D-glucopyrnosyl-(1 ! 4)-[ꢀ-L-arabinopyrano-
syl-(1 ! 6)]-ꢁ-D-glucopyranosid (5) (Ju, Peng, Jia, & Sun, 1994), which were determined
by interpretation of their spectroscopic data as well as by comparison with reported data.

1
258
C. Zhang et al.
All the isolated compounds were evaluated for their cytotoxicity against A549, Hela
and LAC human cancer cell lines following a standard MTT assay. Only compound 1
exhibited a significant cytotoxic activity against all the three cell lines, with IC50 values of
3
.70, 3.64 and 5.70 mM, comparing to the positive control of doxorubicin with IC50 values
of 1.08, 0.95 and 1.16 mM, respectively, while other saponins (2–5) were found to be
inactive (IC 4 100 mM).
5
0
3
3
. Experimental
.1. General
1
13
The H- (500 MHz), C- (125 MHz) and 2D-NMR spectra were determined on a Bruker
DRX-500 instrument using TMS as internal standard. FAB-MS were collected on VG
Auto Spec 3000 FAB/HRMS instrument. Melting points were detected on a Yanagimoto
Seisakusho MD-S2 and uncorrected. Optical rotations were obtained on a Perkin Elmer
3
41 polarimeter with MeOH as solvent. The IR spectra were measured in KBr on an
Equinox spectrophotometer. HRESI–MS data were obtained on an API QSTAR mass
spectrometer. GC analysis was performed on a Thermoquest gas chromatograph using a
2
CP-sil 5 CB capillary column (30 m � 0.25 mm , i.d.), with detection by FID.
Preparative HPLC was run with a Shimazu LC-6 A pump and a Shimazu RID-10 A
2
refractive index detector using an XTerra prep MS C18 column (10 mm, 300 � 19 mm ). For
column chromatography (CC), silica gel (200 ꢀ 300 mesh; Qingdao Puke Isolation
Material Co.), ODS-A (75 mm, YMC) and Sephadex LH-20 (Mitsubishi Chemical
Holdings Co.) were used. Thin-layer chromatography was performed on HSGF254
(
Qingdao Puke Isolation Material Co.) plates.
3
.2. Plant material
The rhizomes and roots of S. scobinicaulis C.H. were collected from Taibai Mountain of
Shaanxi Province, People’s Republic of China, in October 2008. The plant was identified
by Zhenghai Wu of Northwest A&F University. An authenticated voucher specimen (XB
0
0045) was deposited at the Herbarium of Northwest A&F University.
3
.3. Extraction and isolation
The air-dried and powdered plant material (30 kg) was extracted with 70% EtOH at room
temperature and concentrated in vacuo to get a crude extract, which was suspended in H O
2
and partitioned successively with petroleum ether, EtOAc and n-BuOH. The n-BuOH
soluble portion (800 g) was chromatographed on a D101 macroporous adsorption resin and
eluted successively with 40% EtOH, 50% EtOH, 60% EtOH, 70% EtOH and 80% EtOH,
respectively, to afford five fractions Fr. 1ꢀ5. Fr.2 (42 g) was chromatographed over silica
gel CC and eluted with CHCl : MeOH : H O (7 : 3 : 0.5) to yield two subfractions Fr. 2a
3
2
and 2b. Fr. 2a was purified by a reversed-phase (RP-C ) CC and eluted with 70%
1
8
MeOH–H O to give compounds 1 (17 mg) and 5 (26 mg). Fr.3 (83 g) was subjected to a
2
silica gel CC using a gradient of CHCl : MeOH (from 9 : 1 to 8 : 2) to afford two
3
subfractions Fr. 3a and 3b. Further purification of Fr.3b by preparative HPLC with
MeOH–H O (55%) gave compound 4 (17 mg). Fr.5 (54 g) was subjected to a silica gel CC
2
using CHCl : MeOH : H O (8 : 2 : 0.2) to provide three subfractions Fr. 5a, 5b and 5c.
3
2
Fr.5c was purified by preparative HPLC using 50% MeOH–H O to yield compounds 2
2
(
45 mg) and 3 (27 mg).
25 R)-5�-spirostan-3ꢀ,6ꢀ-diol 3-O-ꢀ-D-glucopyranosyl-(1 ! 4)-[�-L-arabinopyrano-
(
syl-(1 ! 6)]-ꢀ-D-glucopyranoside (1) was obtained as white, amorphous power;
ꢁ
20
ꢁ
m.p. 4 280 C; ½�ꢂ ꢀ8 (c 0.5, MeOH).
D

Natural Product Research
1259
1
H-NMR (500 MHz, pyridine-d ): � 0.88 (3H, s, CH -18), 1.13 (3H, s, CH -19), 1.01
5
3
3
(
3H, d, J ¼ 7.5 Hz, CH -21), 0.67 (3H, d, J ¼ 6.5 Hz, CH -27), 3.83 (1H, overlapped, H-3),
3
3
4
.04 (1H, brq, J ¼ 2.9 Hz, H-6), 1.73 (1H, dd, J ¼ 7.5 and 7.5 Hz, H-17), 1.84 (1H, dt,
J ¼ 6.6 and 6.6 Hz, H-20), 3.40 (1H, dd, J ¼ 9.5 and 11.0 Hz, H-26a), 3.59 (1H, brd,
0
J ¼ 10.5 Hz, 1H, H-26b), 4.93(1H, d, J ¼ 7.6 Hz, H-1 ), 3.85 (1H, dd, J ¼ 7.5 and 8.8 Hz,
0
0
0
H-2 ), 4.45 (1H, overlapped, H-3 ) 4.45(1H, dd, J ¼ 9.2 and 8.8 Hz, H-4 ), 3.97 (1H, brd,
0
0
J ¼ 8.2 Hz, H-5 ), 4.82 (1H, dd, J ¼ 8.0 and 13.2 Hz, H-6 a), 4.68 (1H, dd, J ¼ 5.6 and
0
00
00
8
(
1
4
4
.0 Hz, H-6 b), 5.54 (1H, d, J ¼ 7.8 Hz, H-1 ), 4.12 (1H, dd, J ¼ 9.0 and 7.8 Hz, H-2 ), 4.37
1H, dd, J ¼ 9.0 and 9.2 Hz, H-3 ), 4.25 (1H, overlapped, H-4 ), 4.30 (1H, dd, J ¼ 5.5 and
00
00
00
00
000
1.5 Hz, H-6 ), 4.20 (1H, dd, J ¼ 9.0 and 10.0 Hz, H-6 b), 5.11 (1H, d, J ¼ 7.5 Hz, H-1 ),
.56 (1H, dd, J ¼ 10.0 and 7.5 Hz, H-2 ), 4.05 (1H, dd, J ¼ 8.0 and 4.5 Hz, H-C3 ),
.25 (1H, overlapped, H-4 ), 4.24 (1H, overlapped, H-5 a) and 3.73 (1H, brd,
000
000
000
000
000
J ¼ 11.5 Hz, H-5 b).
1
3
C-NMR (125 MHz, pyridine-d ): � 35.3 (C-1), 30.3 (C-2), 77.7 (C-3), 32.8 (C-4), 49.0
5
(
C-5), 71.9 (C-6), 41.0 (C-7), 30.7 (C-8), 54.5 (C-9), 35.9 (C-10), 21.2 (C-11), 40.3 (C-12),
0.8 (C-13), 56.5 (C-14), 21.1 (C-15), 81.2 (C-16), 63.1 (C-17), 16.7 (C-18), 12.4 (C-19), 42.0
C-20), 15.1 (C-21), 109.3 (C-22), 31.8 (C-23), 28.8 (C-24), 30.6 (C-25), 66.9 (C-26), 17.4
4
(
(
(
(
0
0
0
0
0
0
C-27), 102.1 (C-1 ), 74.8 (C-2 ), 76.4 (C-3 ), 81.1 (C-4 ), 74.8 (C-5 ), 68.4 (C-6 ), 104.9
00
00
00
00
00
00
000
C-1 ), 75.2 (C-2 ), 78.5 (C-3 ), 71.8 (C-4 ), 78.4 (C-5 ), 62.5 (C-6 ), 105.6 (C-1 ), 72.6
000
000
000
000
C-2 ), 74.6 (C-3 ), 69.8 (C-4 ) and 67.3 (C-5 ).
�
Negative FABMS: m/z 887 [M � H] (1 0 0); HR–ESI–MS (negative-ion mode) m/z
�
8
87.4638 [M � H] (Calcd C H O , 887.4640).
4
4
71 18
�
1
(KBr) cm : 3421, 2933, 1049, 952, 898 and 863.
IR v
max
3
.4. Acid hydrolysis and sugar analysis
Acid hydrolysis and sugar analysis was performed strictly as previously reported (Zhang,
Gao, & Zhu, 2012). Briefly, a solution of compound 1 (5 mg) was hydrolysed with 2 M
HCl in MeOH–H O (1 : 1) on boiling water at Ar atmosphere. The reaction mixture was
2
determined on a TLC plate with n-BuOH–Me CO–H O (4 : 5 : 1) for sugar analysis. The
2
2
�
1
aqueous residue was treated with pyridine and L-cysteine methyl ester (0.06 mol L
)
hydrochloride for 60 min to obtain a thiazolidine carboxylates, and subsequently silylated
by treatment with hexamethyldisilazane–trimethylchlorosilane (3 : 1). After centrifugation,
the supernatant was concentrated and partitioned between n-hexane and water, and the
hexane phase was subjected to GC. The absolute configurations of the monosaccharides
were confirmed to be L-arabinose and D-glucose by comparison of the retention times
of monosaccharide derivatives with those of standard samples: L-arabinose
(
RT: 10.23 ꢀ 0.01 min) and D-glucose (RT: 14.24 ꢀ 0.01 min), respectively.
3
.5. Cytotoxicity assay
Cytotoxicity was determined against A549 (human lung cancer), LAC (human pulmonary
carcinoma) and Hela (human cervical carcinoma) tumour cell lines based on the MTT
assay method. The stock solution (10 mM) of 1 was prepared by dissolved in 0.5%
dimethylsulphoxide (DMSO), and diluted with culture medium for experimentation. The
tumuor cancer cell lines were cultured in the Dulbecco’s Modified Eagle’s Medium. The
assay procedure was carried out as previously described (Mosmann, 1983), and
doxorubicin was used as the positive control.

1
260
C. Zhang et al.
Acknowledgements
This project was jointly financially supported by the National Natural Science Foundation of China
(
(
30840063, 30770237) and Startup Foundation for Doctors of Northwest A&F University
01140506), as well as the Programme for Changjiang Scholars and Innovative Research Team in
University (IRT0749).
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