New furostanol saponins from Smilax aspera L. and their in vitro cytotoxicity

Article abstract of DOI:10.1016/j.fitote.2010.10.012

The occurrence of the two new cis-fused A/B rings furostanol saponins (25S)-26-O-β-Dglucopyranosyl- 5β-furostan-1β,3β,22α, 26-tetraol-1-O-β-D-glucopyranoside and (25S)-26-O- β-D-glucopyranosyl- 5β-furostan-1β,2β,3β,5β,22α,26-hexaol and the known compounds (25S)-26-O-β-D-glucopyranosyl-5β-furostan-3β, 22α,26-triol-3-O-α-L-rhamnopyranosyl- (1→2)-O-β-D- glucopyranosyl-(1→2)-O-β-D-glucopyranoside and (25S)-26-O-β-D- glucopyranosyl- 5β-furostan-3β,22α,26-triol-3-O-β-D- glucopyranosyl-(1→2)-O-β-D-glucopyranoside, trans-resveratrol, (+) catechin and (-) epicatechin in the rhizomes of Smilax aspera is reported. All saponins have been isolated as their 22-OMe derivatives, which were further subjected to extensive spectroscopic analysis. The isolated furostanol saponins were evaluated for cytotoxic activity against human normal amniotic and human lung carcinoma cell lines using neutral red and MTT assays. In vitro experiments showed significant cytotoxicity in a dose dependent manner with IC₅₀ values in the range of 32.98-94.53 μM.

Full text of DOI:10.1016/j.fitote.2010.10.012

Fitoterapia 82 (2011) 282–287
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
New furostanol saponins from Smilax aspera L. and their in vitro cytotoxicity
a
b
Antoaneta Ivanova a, , Bozhanka Mikhova , Tsvetelina Batsalova ,
⁎
Balik Dzhambazov b, Ivanka Kostova a
a
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Department of Developmental Biology, Biological Faculty, Plovdiv University, 4000 Plovdiv, Bulgaria
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 August 2010
Accepted in revised form 13 October 2010
Available online 21 October 2010
The occurrence of the two new cis-fused A/B rings furostanol saponins (25S)-26-O-β-D-
glucopyranosyl-5β-furostan-1β,3β,22α,26-tetraol-1-O-β-D-glucopyranoside and (25S)-26-O-
β-D-glucopyranosyl-5β-furostan-1β,2β,3β,5β,22α,26-hexaol and the known compounds
(25S)-26-O-β-D-glucopyranosyl-5β-furostan-3β,22α,26-triol-3-O-α-L-rhamnopyranosyl-
(1→2)-O-β-D-glucopyranosyl-(1→2)-O-β-D-glucopyranoside and (25S)-26-O-β-D-glucopyra-
nosyl-5β-furostan-3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1→2)-O-β-D-glucopyranoside,
trans-resveratrol, (+) catechin and (−) epicatechin in the rhizomes of Smilax aspera is
reported. All saponins have been isolated as their 22-OMe derivatives, which were further
subjected to extensive spectroscopic analysis. The isolated furostanol saponins were evaluated
for cytotoxic activity against human normal amniotic and human lung carcinoma cell lines
using neutral red and MTT assays. In vitro experiments showed significant cytotoxicity in a dose
dependent manner with IC50 values in the range of 32.98–94.53 µM.
Keywords:
Smilax aspera
Smilacaceae
Sarsaparilla
Furostanol saponins
Cytotoxic activity
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
formation with cholesterin [4]. During recent years, they have
attracted a growing interest owing to the range of their
Smilax aspera L., familiarly known as sarsaparilla, is an
evergreen, creeping, and extremely tough shrub of the family
Liliaceae, typical of the Mediterranean region. Leaves and
roots of this plant have edible use: the roots are used as
ingredient of soft drink and young shoots can be cooked and
used as an asparagus substitute [1]. S. aspera has also been
used traditionally for treatment of syphilis, diabetes, and
rheumatism, and as an antioxidant and for treatment of
symptoms of menopause in women [2]. S. aspera finds
application in herbal medicine for its depurative, diaphoretic,
diuretic, stimulating and tonic action [1].
biological actions including antidiabetic, antitumor, antitus-
sive, and antidementia and as platelet aggregation inhibitors
[5].
In continuation of our investigations on the chemistry of
Smilax species [6,7], we have now analyzed the chemical
composition of S. aspera rhizomes. In our present study of S.
aspera, growing in France, we found the presence of two new
furostanol saponins (25S)-26-O-β-D-glucopyranosyl-5β-
furostan-1β,3β,22α,26-tetraol-1-O-β-D-glucopyranoside
(1a) and (25S)-26-O-β-D-glucopyranosyl-5β-furostan-
1β,2β,3β,5β,22α,26-hexaol (2a) and five known compounds
(25S)-26-O-β-D-glucopyranosyl-5β-furostan-3β,22α,26-
triol-3-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyrano-
syl-(1→2)-O-β-D-glucopyranoside (3a), (25S)-26-O-β-D-glu-
copyranosyl-5β-furostan-3β,22α,26-triol-3-O-β-D-glucopyr-
anosyl-(1→2)-O-β-D-glucopyranoside (4a), trans-resveratrol
(5), (+) catechin (6) and (−) epicatechin (7). All saponins
have been isolated from the methanol extract of the plant as
their 22-OMe derivatives 1–4. Steroidal saponins (1–4) were
evaluated for their in vitro cytotoxic activity.
The isolation of steroidal saponins, trans-resveratrol and
anthocyanins from S. aspera has been reported [1–3].
The steroidal saponins possess properties such as forth
formation, hemolytic activity, toxicity to fish, and complex
⁎
Corresponding author. Permanent address: Institute of Organic Chemistry
with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia,
Bulgaria. Tel.: +359 2 9606 141; fax: +359 2 87 00 225.
E-mail address: ivanova111@yahoo.com (A. Ivanova).
0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2010.10.012

A. Ivanova et al. / Fitoterapia 82 (2011) 282–287
283
2. Experimental methods
2.4. Acid hydrolysis
2.1. General
A solution of compounds 1 and 2 (1 mg) in 2 M HCl:
dioxan (1:1, 4 ml) was heated at 90 °C for 2 h. The reaction
mixture of each compound was diluted with H2O and then
extracted with CH2Cl2. The water layer was neutralized with
2 M aqueous KOH and then extracted with acetone. The
acetone extract was found to contain glucose by co-TLC
comparison with authentic sample (n-BuOH:pyridine:H2O/
6:4:3) for both investigated compounds.
The NMR experiments were recorded in pyridine-d5 and
CD3OD with TMS as internal standard on a Bruker Avance II+
600 NMR spectrometer, using the standard Bruker software.
HRESIMS was done with a Waters QToF Premier instrument
(Hannover, Germany) with an ESI-ion source equipped with
an Acquity UPLC console. Column chromatography: silica gel
(Merck, 230–400 mesh). Low pressure liquid chromatogra-
phy (LPLC): Lobar pre-packed column LiChroprep RP8
(Merck, 310 mesh). Preparative TLC: silica gel 60 F254
precoated aluminium sheets (0.2 mm layer thickness,
Merck), detection by spraying with 1% solution of vanillin in
concentrated sulfuric acid and Ehrlich reagent (1 g 4-
dimethylaminobenzaldehyde, in a mixture of 25 ml 37%
hydrochloric acid and 75 ml methanol) followed by heating:
the saponins, furostanol form, are coloured bright red.
2.5. Gas chromatographic–mass spectrometry analysis
About 10.0 mg from S5 was dissolved in 60 μl dry pyridine,
35 μl N,O-bis(trimethylsilyl) acetamide was added and the
mixture was heated at 80 °C for 40 min in a screw-cap vial.
The sample was investigated by analytical GC–MS on Hewlett
Packard Gas Chromatograph 6890 equipped with a Hewlett
Packard MS 5973 detector. HP5-MS capillary column
(30 m×0.25 μm film thickness, Agilent Technologies, Wil-
mington, Delaware, USA) was used. The temperature was
2.2. Plant material
programmed from 40 °C to 280 °C at a rate of 6 °C min−1
.
Helium was used as a carrier gas at 9 ml min−1. The ion
source was set at 250 °C and the ionization voltage was 70 eV.
(+) Catechin (2) and (−) epicatechin (3) were identified
using computer searches in the HP Mass spectral Library
NIST98 (Hewlett-Packard, Palo, Alto, California, USA).
The rhizomes of S. aspera L. (Smilacaceae) were collected
in July 2007 near the Observatory of Nice, France. The plant
material was authenticated by Mr. Gerard Gapin, National
Forest Agency, Nice, France.
2.6. Data of compounds
2.6.1. Compound 1
2.3. Extraction and isolation
The air-dried milled rhizomes of S. aspera (650 g) were
extracted three times with methanol at room temperature
for 24 h (3×2500 ml) and filtered. Evaporation of the
solvent under reduced pressure provided the methanol
extract (60.5 g). The extract was suspended H2O/MeOH (1:1,
300 ml) and partitioned with chloroform (3×200 ml) and n-
butanol (3×200 ml). The n-BuOH (saponin fraction, 19.6 g)
was subjected to liquid-vacuum chromatography eluting
with CHCl3:MeOH:H2O (6:1:0.1 to 1:1:0.1) to give 12
fractions (F1–F12). Fraction F3 (158.8 mg) was purified by
silica gel CC eluting with CHCl3:MeOH:H2O (6:1:0.1 to
1:1:0.1) to give six sub-fractions (R1–R6). From R3
(25.0 mg) by preparative normal phase silica TLC (CHCl3:
MeOH:H2O/5:1:0.1, multiple developments) trans-resvera-
trol (5) was obtained. F5 was subjected to silica gel CC
eluting with CHCl3:MeOH:H2O (4:1:0.1 to 1:1:0.1) to give 10
fractions (S1–S10). Fraction S5 was sililated and (+)
catechin (6) and (−) epicatechin (7) were detected by
GC/MS analysis. Fraction F6 (1.1 g) was subjected by silica
gel CC eluting with CHCl3:MeOH:H2O (4:1:0.1 to 1:1:0.1) to
give 10 fractions (M1–M10). From M7 (352.9 mg) com-
pound 1 (14.0 mg) was obtained as crystals. Fraction F8
(0.9 g) was subjected to RP8 LPLC eluting with MeOH:H2O
(3:7 to 4:1, v/v) to give 10 sub-fractions (N1-N10). N3 was
purified on Sephadex LH-20 with methanol and compound 2
(12.2 mg) was obtained. Fraction N6 was purified on
Sephadex LH20 with methanol. Combined fractions contain-
ing furostanol saponins were subjected by CC eluting with
CHCl3:MeOH:H2O (3:1:0.1 to 1:1:0.1) to give 3 (16.0 mg)
and 4 (12.9 mg).
2.6.1.1. Amorphous solid. HRESIMS: Calcd for C40H68O15 (M+
Na): 811.4456. Found: 811.4437. 1 H (d5-pyridine, 600 MHz)
and 13 C (d5-pyridine, 150 MHz), see Table 1.
2.6.2. Compound 2
2.6.2.1. Amorphous solid. HRESIMS: Calcd for C34H58O12 (M+
Na): 681.3826. Found: 681.3870. 1 H (d5-pyridine, 600 MHz)
and 13 C (d5-pyridine, 150 MHz), see Table 2.
2.7. Cell lines and exposure conditions
Two different commercially available cell lines were used for
the in vitro cytotoxicity tests: FL (normal amniotic human cells,
ATCC CCL-62) and A549 (human lung carcinoma, ATCC CCL-
185). Cells were grown in 75 cm2 flasks in Dulbecco's modified
Eagle's medium (DMEM, Gibco™, Paisley, Scotland, UK),
supplemented with 10% (v/v) heat inactivated fetal calf serum
(FCS, PAA Laboratories GmbH, Linz, Austria), 100 U/ml penicil-
lin and 100 μg/ml streptomycin (Sigma, Steinheim, Germany) in
a humidified atmosphere of 5% CO2, 95% air at 37 °C. Prior to
exposure, cells were plated in 96-well tissue culture plates at a
density of 4.5×104cells/ml. After 24 h of attachment, the
cultures were exposed to three concentrations of the tested
compounds dissolved in DMSO—60 µM, 120 µM and 240 µM.
Control wells were prepared by adding equal amounts (20 µL)
of dimethylsulfoxide (DMSO) to the culture medium. The cells
were exposed for 24 h or 48 h prior to analysis. All tests were
performed two times with three replicates per experiment.

284
A. Ivanova et al. / Fitoterapia 82 (2011) 282–287
Table 1
Table 2
1 H (600 MHz) and 13 C NMR (150 MHz) data for 1 in pyridine-d5 (δ in ppm and
J in Hz). (Table 1 shows the 1 H and 13 C NMR data for furostanol saponin 1.)
1 H (600 MHz) and 13 C NMR (150 MHz) data for 2 in pyridine-d5 (δ in ppm
and J in Hz). (Table 2 shows the 1 H and 13 C NMR data for furostanol
saponins 2.)
13 C
79.2
1 H
HMBC C-H
13 C
75.5
1 H
HMBC C-H
1 CH
2 CH2
3 CH
4 CH2
5 CH
6 CH
7 CH2
8 CH
9 CH
10 C
11 CH2
12 CH2
13 C
14 CH
15 CH2
16 CH
17 CH
18 CH3
19 CH3
20 CH
21 CH3
22 C
23 CH2
24 CH2
25 CH
26 CH2
27 CH3
OMe
Glc-1
1′
4.14 brs
2.35 brd (14.5) 1.6
4.34 brs
Me-19, H-1′
a
29.1
66.5
34.5
31.6
26.1
26.3
35.5
41.7
39.4
21.2
40.0
41.1
56.3
32.1
81.3
64.4
16.4
20.0
40.5
16.6
112.6
31.0
28.1
34.5
75.0
17.5
47.3
1 CH
2 CH
3 CH
4 CH2
4.98 brs
Me-19, H-9, H-2
H-1
74.5
69.7
39.6
5.26 brs
5.22 brs
2.60 brd (15.0)
2.17 brd (15.0)
a
2.08 td (13.5, 3.5) 1.6
2.41 m
H-1
H-6, H-1
Me-19
1.3 a, 1.2
a
1.7 a, 1.1
5 C
74.5
35.9
28.7
34.8
45.2
46.0
21.3
39.7
40.8
55.6
32.1
81.2
64.2
16.3
13.6
40.6
16.4
112.6
31.1
28.1
34.4
74.9
17.6
47.4
Me-19, H-9
a
a
a
1.55
1.2
6 CH2
7 CH2
8 CH
9 CH
10 C
11 CH2
12 CH2
13 C
2.0 a, 1.6
a
a
Me-19
Me-19
1.5 a, 0.9
a
1.6
a
a
1.2
1.25
1.6 a, 1.1
Me-18
Me-18, Me-21
Me-18
Me-19, H-9
a
a
2.0 a, 1.4
a
a
1.1
1.6 a, 1.1
Me-18
Me-18
Me-18
2.0 a, 1.4
a
a
4.51 m
1.80 m
0.80 s
1.28 s
2.25 m
1.17 d (7.0)
14 CH
15 CH2
16 CH
17 CH
18 CH3
19 CH3
20 CH
21 CH3
22 C
23 CH2
24 CH2
25 CH
26 CH2
27 CH3
OMe
Glc-26
1′ CH
2′ CH
3′ CH
4′ CH
5′ CH
6′ CH2
1.0
a
Me-21
2.0 a, 1.4
4.57 m
Me-19
Me-21
1.75 t (7.0)
0.81 s
Me-21, Me-18
1.55 s
2.20 m
OMe, Me-21, H-20
2.0 a, 1.8
1.23 d (7.0)
Me-21
OMe
a
1.7 a, 1.4
Me-27
Me-27
Me-27
a
a
a
1.9
2.0 a, 1.9
a
4.08 m 3.52 m
1.04 d (6.7)
3.28 s
1.8 a, 1.5
Me-27
Me-27
Me-27, H-1′
2 H-26
a
1.95
4.14 m 3.57 m
1.1 d (7.1)
3.31 s
101.5
75.4
78.5
5.03 d (7.8)
a
2′
3′
4′
5′
6′
Glc-26
1″
2″
3″
4.1
4.3
a
105.1
75.2
78.6
71.6
78.5
62.8
4.90 d (7.8)
4.12 t (7.8)
a
71.7
4.25
4.0
a
a
78.7
4.3
4.3
4.0
63.0 b
4.59 a, 4.41
a
a
a
a
105.1
75.2
78.6
4.86 d (7.8)
4.63 a, 4.45
a
4.1
4.25
4.25
4.00
a
Overlapped signal.
a
a
4″
5″
6″
72.0
a
78.8
62.8 b
4.6 4.4
a
a
at 540 nm using the ELx800™ Absorbance Microplate Reader
(BioTek). Results were analyzed with the Gen5™ software.
a
Overlapped signal.
Interchangeable signals.
b
2.8.2. MTT assay
The MTT (3-(4′,5′-dimethylthiazol-2′-yl)-2,5-diphenylte-
trazolium bromide; Sigma, St Louis, MO, USA) assay is based
on the capacity of mitochondrial succinyl dehydrogenase to
convert the soluble yellow tetrazolium salt into an insoluble
purple-blue formazan product inside living cells. Briefly, after
the desired time of exposure with the tested compounds,
20 μl of MTT solution (5 mg/ml in PBS) was added directly to
each well and incubated at 37 °C for 3 h. Thereafter, the
supernatant was discarded and after washing with PBS,
0.1 ml of DMSO was added to each well in order to facilitate
solubilization of the formazan product. After 15 min at room
temperature the plates were shaken, and absorbance was
read at 570 nm in a ELx800™ Absorbance Microplate Reader
(BioTek). Results were analyzed with the Gen5™ software.
2.8. Cytotoxicity assays
Cytotoxicity was assessed after 24 or 48 h of exposure by
the neutral red assay or the MTT cell viability assay as
described below.
2.8.1. Neutral red assay
The neutral red assay determines the accumulation of the
neutral red dye in the lysosomes of viable (uninjured) cells
after incubation with test agents. Following exposure to the
tested compounds, cells were incubated for 2 h with neutral
red dye (30 μg/ml) dissolved in serum free medium (DMEM).
Cells were then washed with a formol–calcium solution (1%
anhydrous CaCl2 w/v in 0.4% formaldehyde), which removed
the dead cells. The dye was then extracted from the intact
cells with an acetic acid–ethanol solution (1% glacial acetic
acid in 50% ethanol). The absorbance of the solution was read
3. Results and discussion
The methanol extract of the rhizomes of S. aspera was
suspended in water and extracted with chloroform and n-

A. Ivanova et al. / Fitoterapia 82 (2011) 282–287
285
butanol. The BuOH (saponin) fraction was subjected to
repeated liquid-vacuum chromatography, column chromatog-
raphy on silica gel and Sephadex LH-20, reverse phase low-
pressure liquid chromatography (RP LPLC) and preparative thin
layer chromatography (TLC) to give two newly isolated
furostanol steroidal glycosides (25S)-26-O-β-D-glucopyrano-
syl-22α-methoxy-5β-furostan-1β,3β,22α,26-tetraol-1-O-β-D-
glucopyranoside (1) and (25S)-26-O-β-D-glucopyranosyl-22α-
methoxy-5β-furostan-1β,2β,3β,5β,22α,26-hexaol (2), (Fig. 1).
In addition, five known compounds were isolated and
identified as (25S)-26-O-β-D-glucopyranosyl-22α-methoxy-
5β-furostan-3β,22α,26-triol-3-O-α-L-rhamnopyranosyl-
(1→2)-O-β-D-glucopyranosyl-(1→2)-O-β-D-glucopyranoside
(3), (25S)-26-O-β-D-glucopyranosyl-22α-methoxy-5β-furo-
stan-3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1→2)-O-β-D-
glucopyranoside (4), trans-resveratrol (5), (+) catechin (6)
and (−) epicatechin (7). The 22-Me furostanol saponins 1–4
are artefacts formed during the extraction and isolation
procedure from the corresponding natural 22-OH compounds
(25S)-26-O-β-D-glucopyranosyl-5β-furostan-1β,3β,22α,26-
tetraol-1-O-β-D-glucopyranoside (1a), (25S)-26-O-β-D-gluco-
pyranosyl-5β-furostan-1β,2β,3β,5β,22α,26-hexaol (2a),
(25S)-26-O-β-D-glucopyranosyl-5β-furostan-3β,22α,26-triol-
3-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranosyl-
(1→2)-O-β-D-glucopyranoside (3a) and (25S)-26-O-β-D-
glucopyranosyl-5β-furostan-3β,22α,26-triol-3-O-β-D-glucopyr-
anosyl-(1→2)-O-β-D-glucopyranoside (4a). The easy conversion
of 22-OH furostanol saponins in methanol to their 22-OMe
derivatives is well documented in the literature [8]. The 22-OMe
compounds 1–4 were further used in the structural analysis.
The structures of saponins 1 and 2 were established by
detailed spectroscopic analysis 1D NMR and 2D NMR (1 H,
13 C, COSY, HSQC, HMBC, ROESY) and HRESIMS.
Compound 1 was isolated as an amorphous white solid
and showed positive reaction (red colour) to the Ehrlich
reagent. The pseudo-molecular ion peak [M+Na]+ at m/z
1 R=Me
1a R= H
2 R=Me
2a R=H
3 R=Me
3a R=H
4 R=Me
4a R=H
Fig. 1. Chemical structures of furostanol saponins. (In Figure 1 are given the chemical structures of furostanol saponins 1–4 found in Smilax aspera L.).

286
A. Ivanova et al. / Fitoterapia 82 (2011) 282–287
811.4437 (calculated for C40H68O15Na, 811.4456) in its high-
resolution ESIMS was observed. Combined with the 13 C NMR
spectroscopic data, its molecular formula was determined as
The 13 C spectrum exhibited 34 signals for 5 methyl, 11
methylene, 14 methyne and 4 quaternary carbons (Table 2). In
addition to four methyl groups at δ 13 C 17.6 (δH 1.1), 16.3 (δH
0.81), 16.4 (δH 1.23) and 13.6 (δH 1.55) and a typical furostanol
hemiacetal carbon signal at δ 112.6 ppm were detected. The
above data showed that the compound has a furostanol skeleton.
In the HMBC spectrum the cross peak of the signal of Me-19
(δ 1.55 s) with an oxymethyne signal at δ 75.5 and a quaternary
carbon signal at δ 74.5 inferred two hydroxy groups attached to
C-1 and C-5. The signal of H-1 is a broad singlet at δ 4.98 which is
evidence that this proton is equatorial. In the COSY spectrum a
correlation of H-1 with a proton at δ 5.26 brs, (δ 74.5) is visible,
implying a hydroxyl group at C-2. The H-2 is further connected
to H-3 5.22 brs (δ 13 C 69.7). The shape of the signal of H-3 (5.22,
brs) indicated the α-equatorial position of this proton. The
position of both H2-4 (2.60 brd, J15.0; 2.17 brd, J 15.0) connected
to H-3 was established from the COSY and ROESY spectra. The
HSQC spectrum displays the position of C-4 at 39.6 ppm. From
the ROESY spectrum (Fig. 2) the cis-junction of the rings A and B
and the β-axial position of C1-OH, the β-equatorial position of
C2-OH and the β-axial position of C3-OH could be obtained.
The chemical shift value of Me-27 at δ 1.1 and the
difference of the chemical shifts between H-26 equatorial (δ
3.57) and H-26 axial (δ 4.14) being 0.57 ppm suggest a 25S
configuration [9].
C
40H68O15. The 1 H NMR spectrum of 1 showed the presence of
two anomeric signals at δ 5.03 (d, J 7.8 Hz) and δ 4.86 (d, J 7.8 Hz)
suggesting the presence of two sugar moieties (Table 1). In
addition, signals for the 2 three-proton singlet signals at δ 0.80
and 1.28 indicated the presence of two angular methyl groups, as
well as 2 three-proton doublet signals at δ 1.17 (d, J 7.0 Hz) and
1.04 (d, J 6.7 Hz) assignable to secondary methyl groups of the
steroidal skeleton. Two protons attributable to an oxymethylene
H2-26 at δ 4.08 m and 3.52 m were observed in the 1 H NMR
spectrum (Table 1). The 13 C spectrum showed 40 signals for 5
methyl, 11 methylene, 21 methyne and 3 quaternary carbons.
Two angular methyl groups at δ 16.4 and 20.0, two secondary
methyl groups at δ 16.6 and 17.5, a methylene group (δ 75.0)
linked to an oxygen atom and a hemiacetal carbon signal at δ
112.6 were present in the 13 C spectrum. The above data showed
that the compound has a furostanol skeleton.
The cross peak in the ROESY spectrum of the signal of H-5 (δ
2.41) with the signal of Me-19 (δ 1.28) proved the cis-junction
of the rings A and B. In the HMBC spectrum, the cross peak
between Me-19 at δ 1.28 and the oxymethine signal at δ 79.2
indicated that a hydroxy group is attached to C-1. In the COSY
spectrum, the signal of H-1 was coupled with two signals at δ
2.35 and δ 1.60 of H-2 which were further coupled to a proton at
δ 4.34 (brs), namely H-3. The α-orientation of H-1 was
supported by the ROESY correlation between H-1 and H-9.
The shape of the signals of H-1 and H-3 appeared as brs. The
lack of large axial-axial coupling constants between H-1 and H-
3 was informative about their α-equatorial positions. The NMR
spectral analysis and the comparison with the literature data
[14] identified that compound 1 has a (25S)-26-O-β-D-
glucopyranosyl-furost-1α,3α,22α,26-tetraol-structure.
The chemical shift value of Me-27 at δ 1.04 and the
difference of the chemical shifts between H-26 equatorial (δ
3.52, 1 H) and H-26 axial (δ 4.08, 1 H) being 0.56 ppm suggest
a 25S configuration [9].
Anomeric 1 H/13 C signals of one sugar unit (δ 4.90/δ
105.1) were observed in the NMR spectra. In the HMBC
spectrum, a correlation peak between H-1′ (δ 4.90 d, J 7.8)
and C-26 (δ 74.9) was detected proving that the sugar unit
was linked to C-26. The splitting of the signal of H-1 (7.8 Hz)
proved the β configuration of C-1′. Identification of the sugar
unit was confirmed by acid hydrolysis of compound 2 where
only glucose could be detected by TLC analysis (Rf =0.53).
The 1D and 2D NMR spectral analysis and the comparison
with the literature data of similar compounds [10] identified
compound 2 as (25S)-26-O-β-D-glucopyranosyl-5β-furostan-
1β,2β,3β,5β,22α,26-hexaol. The isolation of 2 implies the
presence of the corresponding natural compound (25S)-26-
O-β-D-glucopyranosyl-5β-furostan-1β,2β,3β,5β,22α,26-hex-
aol (2a) in the rhizomes of S. aspera. The saponin 2a is also a
new compound.
Anomeric 1 H/13 C signals of two sugar units (δ5.03/δ
101.5 and δ4.86/δ 105.1) were observed in the NMR spectra. A
correlation peak between H-1′ and C-1 (δ 5.03 d, J 7.8/δ 79.2)
and H-1″ and C-26 (δ 4.86 d, J 7.8/δ 75.0) in the HMBC
spectrum showed that the sugar units are linked to C-1 and C-
26. Identification of the sugar units was proved by acid
hydrolysis of compound 1 where only glucose could be
detected by TLC analysis (Rf =0.53).
The structures of the known compounds (3–5) were
determined by comparison of their 1 H and 13 C NMR spectral
data with those reported in the literature. The furostanol
The 1D and 2D NMR spectral analysis and the literature
data on related compounds identified compound 1 as (25S)-
26-O-β-D-glucopyranosyl-22α-methoxy-5β-furostan-
1β,3β,22α,26-tetraol-1-O-β-D-glucopyranoside. The isolation
of 1 suggests that the corresponding natural compound
(25S)-26-O-β-D-glucopyranosyl-5β-furostan-1β,3β,22α,26-
tetraol-1-O-β-D-glucopyranoside (1a) is present as a compo-
nent of S. aspera rhizomes. The saponin 1a has not been
reported in the literature so far.
Compound 2 was isolated as amorphous white solid and
showed positive reaction (red colour) to Ehrlich reagent. The
pseudo-molecular ion peak [M+Na]+ at m/z 681.3870 (calcu-
lated for C34H58O12Na, 681.3826) in its high-resolution ESIMS
was observed. Combined with the 13 C NMR spectroscopic data,
Fig. 2. Key ROESY correlations for aglycone of saponin 2. [In Figure 2 are given
the key ROESY (Rotating frame overhause effect spectroscopy) correlations for
furostanol saponins 2].
its molecular formula was determined as C34H58O12
.

A. Ivanova et al. / Fitoterapia 82 (2011) 282–287
287
Table 3
Cytotoxic effects of furostanol saponins on FL and A549 cell lines (after 24 h exposure) expressed as IC50 values. Results are representative of two experiments and
expressed as mean SEM, n=6. (Table 3 shows the cytotoxic effects of furostanol saponins 1–4.)
Cell line
Assay
1
2
3
4
μM
FL
Neutral red
MTT
Neutral red
MTT
80.08 1.02
69.71 0.28
69.80 0.31
62.94 0.05
94.53 0.20
32.98 0.88
52.13 0.62
84.95 1.13
56.72 0.12
56.83 0.73
41.76 0.17
38.43 0.02
35.16 0.89
42.74 0.75
56.00 0.04
63.58 0.33
A549
saponin 3 and trans-resveratrol (5) were found in S. aspera
[2,3]. (+) Catechin (6) and (−) epicatechin (7) were found in
Smilacis chinae [11], while compound 4 was reported in Yucca
gloriosa [12]. This is the first report for the occurrence of 4, 6
and 7 in S. aspera.
Acknowledgements
This work was supported in part by Bulgarian National
Science Fund (Contract X-1601). The authors are indebted to
Mr. Michel Denny, Neufchâteau and Mr. Gerard Gapin,
National Forest Agency, Nice, France for collecting and
supplying of plant material.
Isolated compounds (1–4) were evaluated for their
cytotoxic activity in vitro using human normal amniotic (FL)
and lung carcinoma (A549) cell lines. Two assays were used to
assess the in vitro cytotoxicity: the neutral red assay and the
MTT assay. All four furostanol saponins showed nonspecific
disturbance of membrane structure and functioning of
lysosomes detected by neutral red assay in both, normal FL
and A549 carcinoma cell lines. The observed dose-dependent
cytotoxic effect was confirmed by the MTT assay as well,
where the decrease in cell viability after treatment with
240 µM of the tested compounds reached 12–17% versus
control. The IC50 values against FL and A549 cells after using
neutral red and MTT assays are presented in Table 3. These
values demonstrate that compounds 1 and 3 have increased
sensitivity towards the lung carcinoma cells (A549) compared
with the normal FL cells. Thus, these two saponins could be
investigated further as potential chemotherapy agents for
preferential cytotoxicity on different cancer cell lines.
Compound 3 has been tested only for antifungal activity
against Candida albicans, C. glabrata and C. tropicals, but it was
devoid from such activity [3]. Another furostanol saponin
(protodioscin) isolated from the rhizome of Dioscorea collettii
has been screened for cytotoxicity in vitro using a panel of 60
human cancer cell lines in the National Cancer Institute (USA)
and it was found that this saponin is cytotoxic against most
cell lines from leukemia and solid tumors with a specific
selectivity against several cell lines including the lung
carcinoma A549 [13]. Since no similarities in action of this
compound to the known reference compounds in the
database have been identified, the authors suggested that a
potential novel mechanism of anticancer action is involved
[14]. Therefore, more investigations on the mechanisms of
action of the furostanol saponins on molecular and cellular
levels are needed.
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