Steroidal saponins from the roots of Smilax sp.: Structure and bioactivity

Article abstract of DOI:10.1016/j.steroids.2012.01.009

Phytochemical characterization of a commercial herb sample supplied as Smilax ornata Lem. (sarsaparilla) led to the isolation of five steroidal saponins, including two new furostanol saponins sarsaparilloside B (1) and sarsaparilloside C (2), whose structures were elucidated via a combination of multistage mass spectrometry (MSⁿ), 1D and 2D NMR experiments, and chemical degradation. The previously unreported spectroscopic characterization of sarsaparilloside (3), Δ²⁰⁽²²⁾-sarsaparilloside (4), and parillin (5) is also provided. The antiproliferative activity of the isolated saponins was compared in six human cell lines derived from different tumor types and one of the structures (2) was particularly active against the HT29 colon tumor cell line.

Full text of DOI:10.1016/j.steroids.2012.01.009

Steroids 77 (2012) 504–511
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Steroidal saponins from the roots of Smilax sp.: Structure and bioactivity
Victoria L. Challinor a, Peter G. Parsons b, Sonet Chap a, Eve F. White a, Joanne T. Blanchfield a,
Reginald P. Lehmann c, James J. De Voss a,
⇑
a School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane QLD 4072, Australia
b Queensland Institute of Medical Research, Brisbane QLD 4006, Australia
c Integria Healthcare, Brisbane QLD 4113, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Phytochemical characterization of a commercial herb sample supplied as Smilax ornata Lem. (sarsapa-
rilla) led to the isolation of five steroidal saponins, including two new furostanol saponins sarsaparillo-
side B (1) and sarsaparilloside C (2), whose structures were elucidated via a combination of multistage
mass spectrometry (MSn), 1D and 2D NMR experiments, and chemical degradation. The previously unre-
Received 16 December 2011
Received in revised form 10 January 2012
Accepted 11 January 2012
Available online 21 January 2012
ported spectroscopic characterization of sarsaparilloside (3),
D
20(22)-sarsaparilloside (4), and parillin (5) is
also provided. The antiproliferative activity of the isolated saponins was compared in six human cell lines
derived from different tumor types and one of the structures (2) was particularly active against the HT29
colon tumor cell line.
Keywords:
Smilax
Bioactivity
Sarsaparilla
Saponins
Ó 2012 Elsevier Inc. All rights reserved.
Sarsaparilloside B
Sarsaparilloside C
1. Introduction
characterized Smilax species are S. aspera L. [4–7], S. china L. [8–10],
and S. lebrunii H.Lév. [11–13], which all contain furostanol and spi-
The genus Smilax (family Smilacaceae) comprises approxi-
mately 260 species of climbing vines that are mostly native to
Central and South America. The roots of a number of Smilax species
(all commonly referred to as ‘‘sarsaparilla’’), in particular Smilax
glauca Walter, Smilax rotundifolia L., Smilax aristolochiifolia Mill.,
Smilax regelii Killip & C.V. Morton,1 and Smilax china L., are used
as herbal medicines. Traditional uses of sarsaparilla include the
treatment of rheumatism, rheumatoid arthritis, syphilis, leprosy,
and skin conditions such as psoriasis, as well as a flavoring in root
beer and herbal teas [1,2].
The phytochemistry of the genus Smilax is characterized by an
abundance of steroidal saponins, a class of plant secondary metab-
olite thought to be responsible for the biological activity proposed
for many medicinal herbs. Steroidal saponins exhibit a range of bio-
activities, including cytotoxic, hemolytic, anti-inflammatory, anti-
fungal, and anti-bacterial properties [3]. The most comprehensively
rostanol steroidal glycosides. The isolation of steroidal saponins
and their aglycones has also been reported from S. excelsa L. [14],
S. officinalis Kunth. [15,16], S. medica Schltdl. & Cham. (synonymous
with S. aristolochiifolia Mill.) [17,18], S. menispermoidea A. DC.
[11,19], S. riparia A. DC. [20], and S. sieboldii Miq. [21–23]. Despite
the long history of medicinal use of sarsaparilla [24] and the exten-
sive phytochemical characterization of the above Smilax species,
little is known about the chemical constituents of S. ornata Lem.,1
a member of the genus reported to posses antileprotic activity
[25,26] and used widely in the herbal medicine industry. Very early
literature suggests the presence of saponins and their sapogenins in
S. ornata [27,28], and we thus chose to investigate any steroidal sap-
onins present in a commercial herb sample that was supplied as S.
ornata (but unable to be botanically identified). We chose to per-
form a preliminary screen for antiproliferative activity of the
isolated saponins, as this is a common bioactivity of many steroidal
saponins. In particular, the cytotoxic activity of a number of sapo-
nins from Smilax has been investigated with mixed results
[4,9,29,30].
⇑
Corresponding author. Tel.: +61 7 3365 3825; fax: +61 7 3365 4299.
E-mail address: j.devoss@uq.edu.au (J.J. De Voss).
Recently it has been proposed that S. regelii Killip &C.V. Morton and S. ornata Lem.
In this work we describe the isolation and structure elucidation
of two new furostanol steroidal saponins, sarsaparilloside B (1) and
sarsaparilloside C (2). In addition, the previously unreported
1
are the same species [Ferrufino-Acosta L. Taxonomic revision of the genus Smilax
(Smilacaceae) in Central America and the Caribbean Islands. Willdenowia.
2010;40:227–80]. Both S. regelii and S. ornata have received only limited phytochem-
ical characterization.
spectroscopic characterization of sarsaparilloside (3), D20(22)
sarsaparilloside (4), and parillin (5) is provided.
-
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2012.01.009

V.L. Challinor et al. / Steroids 77 (2012) 504–511
505
919, 903, 757, 739, 595; HRESIMS: m/z 1089.5423 [M + Na]+ (calcd
2. Experimental
for C51H86NaO23, 1089.5452).
2.1. General methods
2.5. 3-O-{[b-
-glucopyranosyl-(25S)-5b-furostane-3b,22,26-triol (sarsaparilloside
C, 2)
D-glucopyranosyl(1 ? 6)]-b-D-glucopyranosyl}-26-O-b-
D
Optical rotations were measured on a JASCO P-2000 polarime-
ter. 1H and 13C NMR spectra were recorded on a Bruker AV750
spectrometer with either the pyridine-d5 signal (dH 8.71 ppm; dC
149.9 ppm) or TMS (in pyridine-d5/D2O, ꢀ9:1 solvent) as internal
standard. Low resolution and multistage mass spectra were re-
corded on a Bruker ESQUIRE HCT instrument (positive and nega-
tive ion ESI). High resolution mass spectra were acquired on a
Bruker MicrOTOF-Q instrument (positive ion ESI) with internal cal-
ibration using Agilent Tune-Mix. Compounds 1–5 were purified by
semipreparative reverse-phase HPLC performed on a Shimadzu
LC-20AT liquid chromatograph (flow rate of 2 mL/min) equipped
with an ELSDLT detector (52 °C, N2 pressure: 200 kPa), column
Amorphous solid; ½a D22
ꢂ
ꢃ 33.0 (c 0.27, CH3OH); 1H (pyridine-d5/
D2O ꢀ9:1, 750 MHz) and 13C NMR (pyridine-d5/D2O ꢀ9:1, 188 MHz)
spectroscopic data, see Tables 1 and 2; positive ion ESIMS: m/z 943,
925, 763, 583; negative ion ESIMS: m/z 919, 757, 595, 433; HRE-
SIMS: m/z 943.4869 [M + Na]+ (calcd for C45H76NaO19, 943.4873).
2.6. Sarsaparilloside (3)
Off-white solid; m.p. 160–162 °C (decomp.); ½a D20
ꢃ 34.9 (c 0.17,
ꢂ
CH3OH); ½a 2D0
ꢂ
ꢃ 31.7 (c 0.14, H2O); Lit. ½a D20
ꢃ 44 (c 0.86, H2O)
ꢂ
oven (40 °C) and a Phenomenex HPLC column (Luna C-18, 5 lm,
[34,35]; 1H (pyridine-d5, 750 MHz) and 13C NMR (pyridine-d5,
188 MHz) spectroscopic data, see Tables 1 and 2; positive ion
ESIMS: m/z 1251, 1233, 1087, 1071, 925, 909; negative ion ESIMS:
m/z 1227, 1081, 1065, 919, 903, 757, 739, 595; HRESIMS: m/z
1251.6008 [M + Na]+ (calcd for C57H96NaO28, 1251.5980).
250 ꢁ 10 mm). Melting points were performed on a melting-point
apparatus (Dr. Tottoli) and are uncorrected.
2.2. Plant material
Dried plant material was supplied and certified by Heinrich
Klenk GmbH & Co KG as S. ornata. A specimen was deposited
(accession number PHARM-110042) at the Medicinal Plant Herbar-
ium, Southern Cross University, Lismore, Australia.
2.7.
D
20(22)-sarsaparilloside (4)
Amorphous solid; ½a D20
ꢂ
ꢃ 15.7 (c 0.06, CH3OH); Lit. ½a D20
ꢃ 36 (c
ꢂ
0.84, CH3OH) [35]; 1H (pyridine-d5/D2O ꢀ9:1, 750 MHz) and 13C
NMR (pyridine-d5/D2O ꢀ9:1, 188 MHz) spectroscopic data, see
Tables 1 and 2; positive ion ESIMS: m/z 1233, 1087, 1071, 925,
763; negative ion ESIMS: m/z 1209, 1063, 1047, 1029, 901, 739;
2.3. Extraction and isolation
HRESIMS: m/z 1233.5918 [M + Na]+ (calcd for C57H94NaO27
,
Powdered roots and rhizome of Smilax sp. (12 g) were extracted
(80% aq. methanol, 120 mL) with sonication (3 ꢁ 10 min). Follow-
ing filtration and removal of the solvent in vacuo, the crude extract
was partially purified by solid phase extraction (Phenomenex Stra-
ta C-18E cartridge) eluting with H2O followed by methanol. The
methanol fraction was concentrated in vacuo, dissolved (90% aque-
ous methanol, 20 mL), filtered and purified by semipreparative
reverse-phase HPLC (gradient of 25–63% aqueous acetonitrile over
35 min). Four different fractions were collected, numbered I–IV in
order of elution. Fractions I, III and IV were pure and corresponded
1233.5875).
2.8. Parillin (5)
Off-white solid; m.p. 240–244 °C (decomp.); Lit. m.p. 220–
223 °C [36]; ½a 2D1
ꢂ
ꢃ 37.1 (c 0.10, CH3OH); ½a D20
ꢃ 35.7 (c 0.06, 80%
ꢂ
aq. EtOH); Lit. ½a D20
ꢂ
ꢃ 64 (c 1.00, 80% aq. EtOH) [36]; 1H (pyri-
dine-d5/D2O ꢀ9:1, 750 MHz) and 13C NMR (pyridine-d5/D2O
ꢀ9:1, 188 MHz) spectroscopic data, see Tables 1 and 2; positive
ion ESIMS: m/z 1071, 925, 909, 763, 601; negative ion ESIMS:
m/z 1083, 1047, 901, 885, 739, 577; HRESIMS: m/z 1071.5333
[M + Na]+ (calcd for C51H84NaO22, 1071.5346).
to sarsaparilloside 3 (Rt: 10.8 min, 80.6 mg),
D
20(22)-sarsaparillo-
side 4 (Rt: 16.4 min, 11.1 mg) and parillin 5 (Rt: 32.7, 16.9 mg).
Fraction II was subjected to further fractionation by semiprepara-
tive reverse-phase HPLC (gradient of 23–28% aqueous acetonitrile
over 45 min) to give two pure fractions that corresponded to sar-
saparilloside B 1 (Rt: 36.8 min, 3.3 mg) and sarsaparilloside C 2
(Rt: 35.3 min, 8.9 mg).
To obtain the aglycone of 1–5, crude extract (from 10 g plant
material) was dissolved in methanol (10 mL) and aqueous HCl
(32%, 1 mL) and heated under reflux for 2 h. After cooling, water
(10 mL) was added and the mixture extracted with diethyl ether
(3 ꢁ 40 mL). The combined organic layers were washed with
aqueous NaOH (5% w/v, 3 ꢁ 40 mL) and dried (MgSO4) before re-
moval of the solvent in vacuo. The residue was purified by flash
chromatography (28% ethyl acetate in hexane, silica gel 60) to yield
sarsasapogenin (7.6 mg), which was identified via comparison of
its 1H and 13C NMR data with literature reports [31–33].
2.9. Determination of sugar absolute configuration
Each saponin (1 mg) was subjected to acid catalyzed methanol-
ysis, before per-trifluoroacetylation of the resultant methylglyco-
sides and enantioselective GC analysis (Chirasil-L-Val capillary
column) according to procedures used previously [37]. The reten-
tion times for the TFAA-derivatized standards were for the two ano-
mers of:
D-glucose (26.21 and 29.97 min);
L
-glucose (26.13 and
29.79 min);
D-rhamnose (17.63 and 23.60 min); and
L
-rhamnose
(17.33 and 23.30 min). The retention times for the TFAA-derivatized
saponin hydrolysates were for: 1 (17.33, 23.31, 26.24 and 29.99
min); 2 (17.31, 23.31, 26.19 and 30.00 min); 3 (17.33, 23.31, 26.20
and 29.99 min); 4 (17.31, 23.30, 26.22 and 29.99 min); and 5
(17.31, 23.31, 26.18 and 29.99 min).
2.4. 3-O-{[
b- -glucopyranosyl}-26-O-b-
3b,22,26-triol (sarsaparilloside B, 1)
a
-
L
-rhamnopyranosyl(1 ? 4)][b-
D-glucopyranosyl(1 ? 6)]-
D
D-glucopyranosyl-(25S)-5b-furostane-
2.10. Inhibition of human cell proliferation assay
Cells seeded at 3000–5000 cells/well in a 96-well plate were
treated with compounds diluted from 10 mg/mL stock solutions
in DMSO, giving a highest final concentration of DMSO (at 50 lg/
mL of compound) of 0.5%. After incubation for 6 days, the plates
were fixed in ethanol and cell content compared with untreated
Amorphous solid; ½a D23
ꢂ
ꢃ 27.0 (c 0.13, CH3OH); 1H (pyridine-d5/
D2O ꢀ9:1, 750 MHz) and 13C NMR (pyridine-d5/D2O ꢀ9:1,
188 MHz) spectroscopic data, see Tables 1 and 2; positive ion
ESIMS: m/z 1089, 1071, 925, 791; negative ion ESIMS: m/z 1065,

506
V.L. Challinor et al. / Steroids 77 (2012) 504–511
Table 1
1H (750 MHz) and 13C (188 MHz) NMR spectroscopic data for the aglycone moiety of 1–5.
1a
2a
3b
4a
5a
1H [d, mult, J (Hz)]
13C
30.9
1H [d, mult, J (Hz)]
13C
31.0
1H [d, mult, J (Hz)]
13C
30.9
1H [d, mult, J (Hz)]
13C
30.8
1H [d, mult, J (Hz)]
13C
30.8
1a
1b
2a
2b
3
4a
4b
5
6a
6b
7a
7b
8
1.48 mc
1.70 mc
1.57 m
1.95 mc
4.43 m
1.82 mc
1.82 mc
2.03 mc
1.15 m
1.76 mc
0.93 m
1.25 mc
1.46 mc
1.26 mc
1.53 m
1.46 mc
1.80 mc
1.46 mc
1.85 m
4.26 mc
1.76 mc
1.76 mc
2.16 m
1.13 m
1.78 mc
0.89 m
1.21 m
1.46 mc
1.23 m
1.49 mc
1.85 mc
1.50 mc
1.89 mc
4.33 mc
1.81 mc
1.81 mc
2.21 mc
1.19 mc
1.83 mc
0.95 m
1.23 mc
1.41 m
1.25 mc
1.47 mc
1.83 mc
1.50 mc
1.89 mc
4.32 mc
1.78 mc
1.78 mc
2.20 m
1.16 mc
1.78 mc
0.92 m
1.22 mc
1.46 mc
1.25 mc
1.75 mc
26.9
1.59 br t (14.2)
27.0
26.8
26.8
26.7
2.04 mc
74.6
30.4
4.46 m
74.6
30.5
75.3
30.7
75.2
30.5
75.2
30.4
1.74 mc
1.82 br t (13.3)
36.9
26.9
2.03 mc
37.0
27.0
36.7
27.0
36.6
26.9
36.4
26.9
1.10 mc
1.75 mc
26.7
0.93 m
26.7
26.8
27.0
26.7
1.26 mc
35.4
40.2
35.2
21.1
1.49 m
35.5
40.2
35.2
21.1
35.5
40.3
35.3
21.2
35.2
40.2
35.2
21.3
35.5
40.2
35.2
21.1
9
1.27 mc
10
11a
11b
12a
12b
13
14
15a
15b
16
17
18
19
20
21
22
23a
23b
24a
24b
25
26a
26b
27
1.19 m
1.20 m
1.33 mc
1.08 mc
1.72 mc
1.18 m
1.29 m
1.06 m
1.69 mc
1.19 mc
1.33 m
1.13 m
1.72 m
1.18 mc
1.30 m
1.06 m
1.68 br d (12.4)
1.32 mc
1.09 br d (13.8)
40.3
40.4
40.4
40.1
40.3
1.73 mc
41.2
56.3
32.3
41.2
56.4
32.4
41.3
56.4
32.5
43.8
54.7
34.4
40.8
56.4
32.1
1.04 mc
1.41 m
2.02 mc
5.02 m
2.02 mc
0.87 s
0.85 s
2.27 m
1.36 d (6.5)
1.06 mc
1.04 mc
0.85 m
1.05 mc
1.42 dt (6.6, 12.7)
1.40 dt (6.2, 12.7)
1.47 mc
2.12 m
1.43 mc
2.03 mc
2.00 mc
2.04 m
81.3
63.6
16.7
23.8
40.5
16.4
110.8
36.9
5.02 td (7.6, 8.3)
2.01 dd (6.6, 8.3)
0.87 s
0.86 s
2.26 m
81.3
63.8
16.7
23.9
40.6
16.4
110.7
37.0
4.98 td (7.1, 7.9)
1.94 mc
0.86 s
0.96 s
2.23 m
81.3
64.1
16.8
24.1
40.7
16.5
110.7
37.2
4.86 mc
2.50 d (10.2)
0.70 s
84.6
64.7
14.4
24.0
103.7
11.8
4.62 mc
81.4
62.8
16.6
24.0
42.5
14.9
109.9
26.3
1.86 dd (6.4, 8.2)
0.83 s
0.97 s
1.94 quin. (6.9)
1.18 d (7.0)
0.99 s
1.36 d (6.8)
1.32 d (6.9)
1.65 s
152.3
23.6
2.08 mc
2.02 mc
1.97 mc
2.20 mc
1.48 mc
1.93 dt (4.9, 13.8)
1.39 m
2.14 m
2.13 dt (4.2, 12.2)
2.08 mc
2.27 m
1.71 mc
28.2
1.72 mc
28.3
1.68 mc
28.4
1.48 mc
31.4
26.1
2.07 mc
2.08 mc
2.05 mc
1.87 mc
2.17 m
1.95 mc
34.3
75.4
1.95 m
34.4
75.4
1.92 mc
34.5
75.4
1.98 m
33.7
75.2
1.62 mc
27.5
65.2
3.53 br t (8.0)
3.51 dd (6.8, 9.4)
4.10 dd (5.7, 9.4)
1.05 d (6.7)
3.48 dd (6.9, 9.4)
4.07 dd (5.8, 9.4)
1.02 d (6.7)
3.52 dd (6.9, 9.4)
4.10 dd (5.7, 9.4)
1.06 d (6.7)
3.42 br d (11.2)
4.11 dd (2.4, 11.2)
1.09 d (7.1)
4.10 mc
1.05 d (6.2)
17.4
17.5
17.5
17.2
16.2
a
b
c
Recorded in pyridine-d5/D2O (ꢀ9:1).
Recorded in pyridine-d5.
Indicates overlapping signals.
controls by staining with sulforhodamine B and quantitation at
540 nm in an ELISA reader. The IC50 values (dose at which cell
growth was inhibited by 50%) were determined by interpolation
on plots of % control absorbance vs dose. For the non-adherent
K562 cell line, MTS was added directly to the cultures after 6 days
of treatment and cell numbers compared with the control after
2–4 h at 37 °C, from the absorbance at 490 nm. Compound 4
caused cell lysis as judged by the appearance of greatly enlarged
cytoplasm. Cancer cell lines were: cervical carcinoma (HeLa); colon
tumor (HT29); breast tumor (MCF7); melanoma (MM96L); and
leukemia (K562).
tion of multistage mass spectrometry (MSn), 1D and 2D NMR spec-
troscopy, and chemical degradation; complete spectroscopic
characterization of 3–5 is reported here.
Compound 1 was isolated as an amorphous solid and positive
ion high resolution (HR) ESIMS provided an ion at m/z
1089.5423, which corresponded to
C
a molecular formula of
51H86O23 (calcd for [M + Na]+: 1089.5452). Fragmentation ob-
served in negative ion ESIMSn showed neutral losses of both 162
and 146 Da from the quasi-molecular ion at m/z 1065 ([M–H]ꢃ),
indicating the presence of both a terminal hexose and deoxyhexose
residue in the saccharide portion of 1. The [M–162–H]ꢃ precursor
ion at m/z 903 was further fragmented to yield neutral losses of
either 146 or 164 Da, which was followed by the loss of 162 Da
from the [M–162–146–H]ꢃ precursor ion. The observed ESIMSn
fragmentation pattern was consistent with a polyhydroxylated
C27 steroidal skeleton accompanied by one deoxyhexose and two
hexose monosaccharides, while the molecular formula for 1 sug-
gested the presence of one additional C6 sugar unit. The 1H NMR
spectrum for 1 (in pyridine-d5/D2O, ꢀ9:1) displayed two signals
typical of the angular methyl group of a steroid at dH 0.85 (s,
H3-19) and 0.87 ppm (s, H3-18), along with two signals at dH 1.05
(d, J = 6.2 Hz, H3-27) and 1.36 ppm (d, J = 6.5 Hz, H3-21) arising
from methyl groups attached to methine carbons. An additional
3. Results and discussion
3.1. Structure elucidation of compounds 1–5
Semipreparative reverse-phase HPLC of the crude methanolic
extract of Smilax sp. underground parts (roots and rhizome) affor-
ded, as a total of 1% of dry weight plant material, two new furost-
anol saponins (1 and 2) and three known compounds (3–5), albeit
ones that had not been spectroscopically characterized previously
(Fig. 1). The extremely limited physical properties reported and the
absence of spectroscopic characterization for sarsaparilloside (3)
methyl signal observed at dH 1.66 (d, J = 5.9 Hz, H3-6, 40-O-
rhamnose) was consistent with the presence of one deoxyhexose
a-L-
[34,35],
necessitated their complete structural elucidation via a combina-
D
20(22)-sarsaparilloside (4) [35], and parillin (5) [34–36]

V.L. Challinor et al. / Steroids 77 (2012) 504–511
507
Table 2
1H (750 MHz) and 13C (188 MHz) NMR spectroscopic data for the sugar portion of 1–5.
1a
2a
3b
4a
5a
1H [d, mult, J (Hz)]
13C
1H [d, mult, J (Hz)]
13C
1H [d, mult, J (Hz)]
13C
1H [d, mult, J (Hz)]
13C
1H [d, mult, J (Hz)]
13C
3–O–b–
D
-Glucose
3–O–b–
4.90 d (7.8)
4.03 t (8.4)
4.24 t (8.9)
4.16 t (9.4)
4.12 m
4.37 dd (6.3, 11.5)
4.83 br d (11.2)
D
-Glucose
3–O–b–
D
-Glucose
3–O–b–
4.84 d (7.6)
4.21 t (8.6)
4.17 t (8.6)
4.23 t (9.2)
3.77 m
4.08 dd (3.9, 11.0)
4.56 br d (11.1)
D
-Glucose
3–O–b–D-Glucose
4.84 d (7.8)
4.21 t (8.9)
4.16 t (8.9)
4.20 t (9.1)
3.76 m
4.07 dd (4.9, 11.2)
4.56 br d (11.2)
10
4.87 d (7.8)
3.95 t (8.2)
4.13 t (8.6)
4.29 t (9.1)
3.83 m
102.6
74.9
76.5
79.3
75.2
68.4
103.0
75.0
78.2
71.5
77.2
70.0
4.78 d (7.5)
101.6
81.9
76.4
78.9
75.3
68.6
101.3
81.3
76.4
78.7
75.2
68.4
101.1
81.0
76.4
78.6
75.2
68.3
20
4.16 mc
30
4.16 mc
40
4.23 t (9.0)
3.75 m
4.07 dd (5.0, 11.2)
4.52 br d (11.4)
50
6a0
6b0
4.12 mc
4.60 br d (10.8)
20–O–b–
D-Glucose
20–O–b–
D-Glucose
20–O–b–
D-Glucose
1
2
3
4
5
6a
6b
5.39 d (7.7)
4.04 t (8.1)
4.25 t (8.7)
4.28 t (8.9)
3.96 m
105.5
77.0
78.0
72.0
78.7
63.0
5.42 d (7.4)
4.05 t (8.0)
4.26 t (8.3)
4.24 t (8.7)
3.98 m
105.0
76.6
77.8
71.8
78.6
62.9
5.43 d (7.7)
4.04 t (8.4)
4.26 t (9.1)
4.21 t (9.1)
3.98 ddd (2.7, 5.5, 9.5)
4.43 dd (5.5, 11.7)
4.60 br d (11.6)
104.8
76.4
77.7
71.8
78.5
62.8
4.47 dd (4.9, 11.4)
4.57 br d (11.4)
4.46 dd (4.8, 10.8)
4.61 br d (11.1)
40–O–
a
–
L-Rhamnose
40–O–
a
–L-Rhamnose
40–O–
a
–
L-Rhamnose
40–O–
a–L-Rhamnose
1
2
3
4
5
6
5.78 br s
4.71 br s
4.55 br d (9.7)
4.34 t (9.3)
4.82 m
102.6
72.2
72.3
73.5
70.5
18.4
5.79 br s
4.68 br s
4.48 dd (2.8, 9.2)
4.29 t (9.3)
4.78 m
102.8
72.5
72.7
73.8
70.5
18.5
5.76 br s
4.69 br s
4.51 dd (2.8, 9.4)
4.33 t (9.4)
4.78 m
102.6
72.2
72.4
73.6
70.5
18.4
5.72 br s
4.67 br s
4.50 dd (3.2, 9.3)
4.32 t (9.4)
4.76 m
102.5
72.1
72.3
73.5
70.4
18.3
1.66 d (5.9)
1.60 d (6.2)
1.62 d (6.2)
1.62 d (6.2)
60–O–b–
D-Glucose
60–O–b–
D-Glucose
60–O–b–
D-Glucose
60–O–b–
D-Glucose
60–O–b–
D-Glucose
1
2
3
4
5
6a
6b
4.98 d (7.8)
4.03 t (8.3)
4.26 t (9.1)
4.15 t (9.0)
3.92 m
104.6
74.6
77.9
71.3
78.1
62.4
5.16 d (7.8)
4.07 t (8.4)
4.26 t (8.9)
4.21 t (9.0)
3.96 m
105.1
75.0
78.2
71.5
78.3
62.6
4.94 d (7.7)
4.02 t (8.4)
4.19 t (9.0)
4.20 t (9.0)
3.85 m
105.0
74.9
78.5
71.5
78.5
62.6
4.96 d (7.8)
4.04 t (8.3)
4.24 t (9.0)
4.19 t (8.9)
3.90 m
104.7
74.7
78.1
71.3
78.3
62.5
4.94 d (7.8)
4.02 t (8.4)
4.24 t (9.1)
4.15 t (9.1)
3.98 ddd (2.3, 5.5, 9.5)
4.29 dd (5.7, 11.8)
4.49 br d (11.8)
104.6
74.6
78.0
71.3
78.2
62.4
4.29 dd (7.1, 12.0)
4.50 br d (12.0)
4.35 dd (5.9, 11.9)
4.52 br d (11.8)
4.33 dd (5.6, 11.7)
4.48 br d (11.7)
4.33 dd (5.6, 11.7)
4.51 br d (11.7)
26–O–b–
D-Glucose
26–O–b–
D
-Glucose
26–O–b–D-Glucose
26–O–b–D-Glucose
1
2
3
4
5
6a
6b
4.81 d (7.7)
4.03 t (8.4)
4.28 t (9.0)
4.18 t (9.2)
3.96 m
104.8
74.9
78.1
71.5
78.1
62.5
4.81 d (7.8)
4.03 t (8.4)
4.27 t (9.0)
4.21 t (9.1)
3.96 m
105.0
75.0
78.3
71.5
78.3
62.6
4.80 d (7.8)
4.02 t (8.3)
4.23 mc
105.2
75.3
78.7
71.7
78.5
62.9
4.85 d (7.8)
4.05 t (8.5)
4.28 t (8.8)
4.21 t (9.3)
3.98 m
105.0
75.0
78.4
71.6
78.4
62.7
4.23 mc
3.93 m
4.38 dd (5.3, 11.8)
4.54 br d (11.8)
4.33 dd (5.8, 11.4)
4.53 br d (12.2)
4.36 dd (6.1, 12.0)
4.54 br d (12.0)
4.37 dd (4.2, 11.6)
4.56 br d (11.4)
a
b
c
Recorded in pyridine-d5/D2O (ꢀ9:1).
Recorded in pyridine-d5.
Indicates overlapping signals.
Fig. 1. Steroidal saponins 1–5 isolated from the roots of Smilax sp.
monosaccharide. The 13C NMR spectrum of 1 contained 51 signals,
which included five methyl groups along with 14 methylene, 29
methine, and only three quaternary carbons, at dC 35.2 (C-10),
41.2 (C-13), and 110.8 ppm (C-22). Correlations observed in the
HSQC and HMBC spectra of 1 allowed assignment of the steroid
methyl groups as dC 16.4 (C-21), 16.7 (C-18), 17.4 (C-27) and
23.8 ppm (C-19). The HMBC correlations of these characteristic
methyl groups allowed the rapid assignment of the steroidal skel-
eton. For example, correlations observed for H3-21 (dH 1.36 ppm)
revealed the signals of C-17 (dC 63.6), C-20 (dC 40.5), and C-22 (dC
110.8), with the chemical shift of C-22 clearly indicating the pres-
ence of a hemiacetal moiety, characteristic of furostanol saponins
[38]. Further HMBC correlations from H3-27 (dH 1.05 ppm) allowed
identification of C-24 (dC 28.2), C-25 (dC 34.3), and C-26 (dC 75.4),
with the downfield shift of the latter suggesting O-glycosylation
at C-26. Examination of TOCSY, COSY, HSQC, and HMBC spectra

508
V.L. Challinor et al. / Steroids 77 (2012) 504–511
allowed the complete 1H and 13C NMR assignment of the steroidal
skeleton of 1 and its identification as a saturated furostanol core
(Table 1). The downfield shift of C-19 in 1 (to dC 23.8 ppm), when
present in 2. Long-range HMBC correlations between dH 4.90
(H-10, 3-O-b-
-glucose) and dC 74.6 (C-3, aglycone), dH 5.16 (H-1,
60-O-b-
-glucose) and dC 70.0 (C-6, 3-O-b- -glucose), and dH 4.81
(H-1, 26-O-b- -glucose) and dC 75.4 (C-26, aglycone) revealed a
D
D
D
compared with its value in analogous H-5
a
furostanes (dC
D
12.3 0.1 ppm) [38], suggested the cis fusion of the steroid A and
B rings. The b orientation of H-5 was confirmed by an intense cross
peak between H3-19 and H-5 in the ROESY spectrum of 1. The
absolute configuration of C-25 in 1 was assigned on the basis of
the geminal proton resonances of H2-26 in pyridine-d5/D2O
1 ? 6 linked diglucose moiety attached at C-3 of the aglycone,
along with a single glucose residue attached at C-26 (Table 2).
The upfield shift of C-40 in 2 (dC 71.5 ppm), when compared
with its chemical shift in 1 (dC 79.3 ppm), confirmed that the C-40
linked rhamnose present in 1 was absent in 2. The structure of 2
(ꢀ9:1) (dH 3.53 and 4.10 ppm,
agreement with the 25S d P 0.57 ppm) rather than 25R
d 6 0.48 ppm) configuration [39]. The orientation of the C-22
D
d = 0.57 ppm), which were in
was therefore elucidated as 3-O-{[b-
D-glucopyranosyl(1 ? 6)]-b-D-
(D
glucopyranosyl}-26-O-b- -glucopyranosyl-(25S)-5b-furostane-3b,
D
(D
a
22,26-triol (sarsaparilloside C). While 2 has not been previously iso-
lated as a natural product, it has been reported in the patent litera-
ture with very limited characterization as a synthetic compound
[41]. 1H and 13C NMR data were available (in pyridine-d5) for the
three anomeric positions only, but the reported chemical shifts
(dH 5.19, 5.28, and 5.31 ppm; dC 102.1, 103.5, and 105.1 ppm) [41]
clearly do not match those for 2; this discrepancy remains to be
resolved.
hydroxyl group was indicated by ROESY correlations between
H-20 (dH 2.27 ppm) and both H-23a (dH 2.08 ppm) and H-23b
(dH 2.14 ppm). The aglycone of 1 was obtained via semipreparative
reverse-phase HPLC performed on the acid hydrolysate of a crude
Smilax sp. extract, and displayed identical spectroscopic (1H and
13C NMR) properties to literature reports for sarsasapogenin
[31–33], in agreement with the structure and stereochemistry
proposed for 1 on the basis of spectroscopic data.
Compound 3 was isolated as an off-white solid, and positive ion
HRESIMS provided an ion m/z 1251.6008, corresponding to a
molecular formula of C57H96O28 (calcd for [M + Na]+: 1251.5980).
Fragmentation in negative ion ESIMSn of the quasi-molecular ion
at m/z 1227 ([M–H]ꢃ) yielded neutral losses of both 146 and
162 Da (m/z 1081 and 1065, respectively), suggesting the presence
of both a terminal deoxyhexose and a hexose residue in a branched
sugar chain. The [M–162–H]ꢃ fragment was selected as a precursor
ion and went onto give successive neutral losses of 162, 164, and
142 Da, consistent with the presence of at least one deoxyhexose
and three hexose monosaccharides; the molecular formula for 3
indicated the presence of an additional C6 sugar. The 1H NMR spec-
trum of 3 (in pyridine-d5) displayed signals for two methyl groups
attached to quaternary carbons, at dH 0.86 (s, H3-18) and 0.96 ppm
(s, H3-19), and two methyl groups attached to methine carbons, at
dH 1.02 (d, J = 6.7 Hz, H3-27) and 1.32 ppm (d, J = 6.9 Hz, H3-21). An
additional methyl doublet signal was observed at dH 1.60 ppm (d,
The 1H NMR spectrum of 1 also contained four signals typical of
the anomeric proton of a glycoside at dH 4.81 (d, J = 7.7 Hz, H-1,
26-O-b-
(d, J = 7.8 Hz, H-1, 60-O-b-
O- -rhamnose) (Table 2). These were correlated in the HSQC
spectrum of 1 with signals at dC 104.8 (C-1, 26-O-b- -glucose),
102.6 (C-10, 3-O-b- -glucose), 104.6 (C-1, 60-O-b-
-glucose) and
102.6 ppm (C-1, 40-O-
-rhamnose), respectively. Examination of
the vicinal coupling constants obtained through selective 1D
TOCSY experiments identified the sugar units of as one
-rhamnopyranosyl and three b-glucopyranosyl residues. The
D
-glucose), 4.87 (d, J = 7.8 Hz, H-10, 3-O-b-
D
-glucose), 4.98
D
-glucose) and 5.78 ppm (br s, H-1, 40-
a-L
D
D
D
a-L
1
a
absolute configuration of the sugar units was determined by enan-
tioselective GC analysis, in which acid catalyzed methanolysis of 1
was followed by per-trifluoroacetylation of the resultant methyl-
glycosides [40]. Comparison of the retention times of authentic,
TFAA-derivatized standards and co-injection of the saponin 1
hydrolysate confirmed the presence of three units of
and one unit of -rhamnose. The sugar linkage pattern was eluci-
dated using correlations observed in the HMBC spectrum of 1. Cor-
relations between dH 4.87 (H-10, 3-O-b-
-glucose) and dC 74.6 (C-3,
aglycone), dH 5.78 (H-1, 40-O- -rhamnose) and dC 79.3 (C-40, 3-O-
b-
-glucose), and dH 4.98 (H-1, 60-O-b- -glucose) and dC 68.4 (C-60,
3-O-b- -glucose) indicated the presence of a branched trisaccha-
ride moiety linked at position C-3 of the aglycone. An additional
correlation between dH 4.81 (H-1, 26-O-b- -glucose) and dC 75.4
D-glucose
J = 6.2 Hz, H3-6, 40-O-
a-L-rhamnose), consistent with the presence
L
of one deoxyhexose monosaccharide. These five methyl group sig-
nals were correlated in the HSQC spectrum of 3 with signals at dC
16.8 (C-18), 24.1 (C-19), 17.5 (C-27), 16.5 (C-21), and 18.5 ppm
D
a
-L
(C-6, 40-O-
a-L-rhamnose), respectively, with the downfield chemi-
D
D
cal shift of C-19 being indicative of a cis A/B ring junction. The
HMBC correlations of the steroid methyl groups provided impor-
tant structural information; the chemical shift of C-22 (dC
110.7 ppm) as revealed by a correlation from H3-21 (dH 1.32 ppm)
was typical of a 22-hydroxy furostanol, while the shift of C-26 (dC
75.4 ppm) was suggestive of 26-O-glycosylation [38]. Complete
1H and 13C NMR assignment of the aglycone of 3 (Table 1) con-
firmed that this saponin, like 1 and 2, is based on sarsasapogenin.
The 1H NMR spectrum of 3 also displayed five anomeric signals,
D
D
(C-26, aglycone) revealed the attachment of a single glucose
residue at position C-26 of the aglycone. The structure of 1 was
therefore identified as 3-O-{[
a-
L
-rhamnopyrnosyl(1 ? 4)][b-
D-glu-
copyranosyl(1 ? 6)]-b- -glucopyranosyl}-26-O-b-
D
D
-glucopyrano-
syl-(25S)-5b-furostane-3b,22,26-triol (sarsaparilloside B).
Compound 2 was isolated as an amorphous solid. Positive ion
HRESIMS provided an ion at m/z 943.4869, consistent with a molec-
ular formula of C45H76O19 (calcd for [M + Na]+: 943.4873). The frag-
mentation pattern observed in negative ion ESIMSn suggested the
presence of three hexose monosaccharides, with the successive
neutral loss of three 162 Da units from the quasi-molecular ion at
m/z 919 ([M–H]ꢃ). Along with the molecular formula for 2, this sug-
gested a structure similar to 1, lacking the deoxyhexose residue.
Accordingly, the 1H and 13C NMR spectroscopic data for the agly-
cone of 2 (in pyridine-d5/D2O, ꢀ9:1) were very similar to those of
1 (Table 1). The 1H NMR spectrum of 2 displayed only three signals
typical of the anomeric proton of a glycoside, at dH 4.81 (d,
at dH 4.78 (d, J = 7.5 Hz, H-10, 3-O-b-
1, 26-O-b-
-glucose), 4.94 (d, J = 7.7 Hz, H-1, 60-O-b-
(d, J = 7.7 Hz, H-1, 20-O-b- -glucose), and 5.79 (br s, H-1, 40-O-
rhamnose) (Table 2). These were correlated in the HSQC spectrum
of 3 with signals at dC 101.6 (C-10, 3-O-b-
-glucose), 102.8 (C-1,
40-O- -rhamnose), 105.0 (C-1, 60-O-b-
-glucose), 105.2 (C-1, 26-
O-b- -glucose). Examina-
-glucose), and 105.5 ppm (C-1, 20-O-b-
tion of 1D TOCSY subspectra for the five individual sugar units,
along with enantioselective GC analysis of the saponin hydrolysate,
led to identification of the sugars of the saccharide portion of 3 as
D
-glucose), 4.80 (d, J = 7.8 Hz, H-
D
D-glucose), 5.39
D
a-L-
D
a
D
-L
D
D
one
The sugar linkage pattern was established through HMBC correla-
tions observed between dH 4.78 (H-10, 3-O-b-
-glucose) and dC
75.3 (C-3, aglycone), dH 5.39 (H-1, 20-O-b-
-glucose) and dC 81.9
(C-20, 3-O-b- -glucose), dH 5.79 (H-1, 40-O-
-rhamnose) and dC
78.9 (C-40, 3-O-b- -glucose), dH 4.94 (H-1, 60-O-b-
-glucose) and
a-L-rhamnopyranosyl and four b-D-glucopyranosyl residues.
J = 7.8 Hz, H-1, 26-O-b-
-glucose), and 5.16 ppm (d, J = 7.8 Hz, H-1, 60-O-b-
Examination of selective 1D TOCSY spectra and enantioselective
GC analysis confirmed that three b- -glucopyranosyl units were
D
-glucose), 4.90 (d, J = 7.8 Hz, H-10, 3-O-b-
-glucose).
D
D
D
D
D
a-L
D
D
D

V.L. Challinor et al. / Steroids 77 (2012) 504–511
509
Table 3
dC 68.6 (C-60, 3-O-b-
D-glucose), and dH 4.80 (H-1, 26-O-b-D-glucose)
IC50 of the isolated saponins 1–5 for inhibition of human cell proliferation against
and dC 75.4 (C-26, aglycone). Compound 3 was thus identified as
sarsaparilloside, a bidesmodic furostanol saponin possessing a
highly branched tetrasaccharide moiety linked at position C-3
[34,35]. While sarsaparilloside (3) has been isolated previously
from S. aristolochiifolia, as part of a mixture containing ‘‘up to
15%’’ of 22-methoxy sarsaparilloside [34,35], its spectroscopic
characterization has not been reported. The optical rotation re-
normal fibroblasts (NFF) and different cancer cell lines (lg/mL).
Compound
Cell line
NFF
HeLa
HT29
MCF7
MM96L
K562
1
2
3
4
5
>50
27
13
4.5
>50
>50
42
12
40
>50
>50
4.8
5
14
>50
>50
24
9.5
3.4
>50
>50
23
14
3.8
>50
>50
28
22
4.3
>50
ported for sarsaparilloside (½a D20
ꢃ 44, c 0.86, H2O) [34,35] (the only
ꢂ
reported physical property) was in fair agreement with that of 3
(½a 2D0
ꢂ
ꢃ 31.7, c 0.14, H2O). The complete 1H and 13C NMR assign-
162 Da (m/z 885), suggesting the presence of a branched polysac-
charide as seen in 3 and 4. The [M–146–H]ꢃ product ion was fur-
ther fragmented to yield two successive neutral losses of 162 Da,
consistent with the presence of at least one deoxyhexose and
two hexose monosaccharides; the molecular formula for 5 indi-
cated the presence of a fourth C6 sugar. The 1H NMR spectrum of
5 (in pyridine-d5/D2O, ꢀ9:1) contained signals for two methyl
groups attached to quaternary carbons, at dH 0.83 (s, H3-18) and
0.97 ppm (s, H3-19), and three methyl groups attached to methine
carbons, at dH 1.09 (d, J = 7.1 Hz, H3-27), 1.18 (d, J = 7.0 Hz, H3-21),
ment of sarsaparilloside (3) (in pyridine-d5) is reported here for
the first time (Tables 1 and 2).
Compound 4 was isolated as an amorphous solid, and positive
ion HRESIMS gave a molecular formula of C57H94O27, from an ion
observed at m/z 1233.5918 (calcd for [M + Na]+: 1233.5875). The
fragmentation pattern observed in negative ion ESIMSn for 4 was
similar to that of 3, with the quasi-molecular ion at m/z 1209
undergoing sequential neutral losses of one 146 Da and three
162 Da units. The 1H NMR spectrum of 4 (in pyridine-d5/D2O,
ꢀ9:1) displayed signals for two methyl groups attached to quater-
nary carbons at dH 0.70 (s, H3-18) and 0.99 ppm (s, H3-19), along
with two signals for methyl groups attached to methine carbons
at dH 1.06 (d, J = 6.7 Hz, H3-27) and 1.62 ppm (d, J = 6.2 Hz, H3-6,
and 1.62 ppm (d, J = 6.2 Hz, H3-6, 40-O- -rhamnose). The 13C NMR
a-L
chemical shifts of these methyl groups were assigned via HSQC as
dC 16.6 (C-18), 24.0 (C-19), 16.2 (C-27), 14.9 (C-21), and 18.3 ppm
(C-6, 40-O-
a-L-rhamnose), with the chemical shift of C-19 again
40-O-
a-L-rhamnose) (Table 1). The methyl doublet signal observed
indicative of the b orientation of H-5. Correlations observed in
the HMBC spectrum of 5 from H3-21 (dH 1.18 ppm) and H3-27
(dH 1.09 ppm) allowed the assignment of positions 22 (dC
109.9 ppm), 24 (dH 1.39 and 2.17 ppm; dC 26.1 ppm), 25 (dH
1.62 ppm; dC 27.5 ppm), and 26 (dH 3.42 and 4.11 ppm; dC
65.2 ppm), revealing F ring chemical shifts typical of a 25S-config-
ured spirostanol [44,45]. Examination of COSY, TOCSY, HSQC, and
HMBC spectra, and enantioselective GC analysis, revealed that 5
contains the same branched tetrasaccharide moiety as found in 3
and 4. Compound 5 was thus identified as parillin, the spirostanolic
analogue of sarsaparilloside (3), first reported along with
compound 3 from S. aristolochiifolia [34–36]. The limited physical
for H3-21 in 3 was absent, but a new methyl singlet was observed
at dH 1.65 ppm. The methyl group signals seen in the 1H NMR spec-
trum of 4 showed HSQC correlations with signals at dC 14.4 (C-18),
24.0 (C-19), 17.2 (C-27), 18.4 (C-6, 40-O-
a-L-rhamnose), and
11.8 ppm (C-21). The long-range HMBC correlations of H3-21 (dH
1.65 ppm) revealed one methine carbon at dC 64.7 ppm (C-17),
which was correlated in the HSQC spectrum with a signal at dH
2.50 ppm (d, J = 10.2 Hz, H-17), as well as two quaternary carbons
at dC 103.7 (C-20) and 152.3 ppm (C-22) in the olefinic region. The
chemical shifts of C-20, C-21, and C-22, and the multiplicity of the
H-17 and H3-21 resonances, suggested a
D
20(22)-unsaturated agly-
cone for 4 and were in agreement with literature values for D20(22)
-
properties reported for parillin (m.p. 220–223 °C; ½a D20
ꢃ 64, c
ꢂ
furostanol saponins derived from sarsasapogenin [42,43].
1.00, 80% aq. EtOH) [36] were in fair agreement with those of 5
The 1H NMR spectrum of 4 contained five signals corresponding
to the anomeric protons of glycosides, at dH 4.84 (d, J = 7.6 Hz, H-
(m.p. 240–244 °C, decomp.; (½a D21
ꢃ 35.7, c 0.06, 80% aq. EtOH).
ꢂ
The previously unreported 1H and 13C NMR assignment of 5 is pro-
10, 3-O-b-
4.96 (d, J = 7.8 Hz, H-1, 60-O-b-
20-O-b- -glucose), and 5.76 ppm (br s, H-1, 40-O-
which displayed HSQC correlations with signals at dC 101.3 (C-10,
3-O-b- -glucose), 105.0 (C-1, 26-O-b-
-glucose), 104.7 (C-1, 60-O-
b- -glucose), and 102.6 ppm (C-1,
-glucose), 105.0 (C-1, 20-O-b-
40-O-
-rhamnose), respectively (Table 2). Careful examination
D
-glucose), 4.85 (d, J = 7.8 Hz, H-1, 26-O-b-
-glucose), 5.42 (d, J = 7.4 Hz, H-1,
-rhamnose),
D-glucose),
vided in Tables 1 and 2.
D
D
a-L
3.2. Biological evaluation
D
D
The isolation of a structurally related series of saponins, differing
in aglycone structure and glycosylation pattern, provides a means
to systematically probe structure–activity relationships of the com-
ponents of this widely used medicinal herb. The prolonged treat-
ment time of sparsely-seeded cultures allowed for comparison of
clonogenic survival of treated cells. Three of the five isolated sapo-
D
D
a-L
of TOCSY, COSY, HSQC, and HMBC spectra for 4 revealed that this
saponin shares the same glycosylation pattern as present in sar-
saparilloside (3). Compound 4 was therefore was identified as
D
20(22)-sarsaparilloside, a 20(22)-unsaturated furostanol saponin
D
nins showed cytotoxic activity in cultured cells at <10 lg/mL, a
previously synthesized from sarsaparilloside (3) and reported with
only limited characterization (optical rotation and melting point)
range with potential for treatment in vivo. Compounds 2 and 3
selectively inhibited the proliferation of the HT29 colon tumor cell
line, whereas 4 was more generally active across the six cell lines
studied (Table 3). Interestingly, HT29 is not particularly sensitive
to other classes of saponin [46,47]. The variation in selectivity for
different tumor types suggests a mechanism more specific than
lysis of the cell membrane. Many studies have demonstrated that
saponins induce apoptosis in tumor cells by a variety of pathways
[48] but few specific ligands have been identified.
[35]. The optical rotation of 4 (½a D20
ꢂ
ꢃ 15.7, c = 0.06, CH3OH) was
in only fair agreement with that reported for
D
20(22)-sarsaparillo-
side (½a 2D0
ꢃ 36 (c = 0.84, CH3OH) [35]; whether this is due to the
ꢂ
difference in concentration or other factors is unknown. We pro-
vide here the complete 1H and 13C NMR assignment of 4 (Tables
1 and 2), a steroidal saponin that has not been previously isolated
from a natural source.
Compound 5 was isolated as an off-white solid and HRESIMS
provided an ion at m/z 1071.5333, consistent with a molecular for-
mula of C51H84O22 (calcd for [M + Na]+: 1071.5346). Fragmentation
in negative ion ESIMSn of the quasi-molecular ion at m/z 1047
([M–H]ꢃ) yielded neutral losses of both 146 Da (m/z 901) and
4. Conclusions
Phytochemical investigation of a commercial herb sample sup-
plied as S. ornata (but unable to be botanically identified) led to the

510
V.L. Challinor et al. / Steroids 77 (2012) 504–511
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nins represent an interesting and apparently almost complete bio-
synthetic pathway, allowing us to propose a route for their
biosynthesis. The presence of a glucose residue at position C-3 in
all of the isolated saponins indicates that this glycosylation step
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larly, C-26 glucosylation is chemically logical to permit formation
of the hemiacetal at position C-22 that can later close to give the
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The complex taxonomy of the genus Smilax makes it difficult to
comment on the chemotaxonomy of this group. The isolated
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the sarsasapogenin aglycone, which is characterized by 25S stereo-
chemistry and the presence of a cis A/B ring junction. Compounds 3
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D
5(6)-unsaturation are found mainly
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The authors wish to thank L. Lambert (Centre for Advanced
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