A new cytotoxic triterpene saponin from Lysimachia nummularia L.

Article abstract of DOI:10.1016/j.carres.2013.04.005

A new glycosylated triterpene 1 (named nummularoside) was isolated from the underground parts of Lysimachia nummularia L. Its chemical structure was elucidated as 3-O-β-{{[β-d-xylopyranosyl-(1→2)]-[β-d- xylopyranosyl-(1→4)]-β-d-glucopyranosyl-(1→4)} -[β-d-glucopyranosyl-(1→2)-]-α-l-arabinopyranosyl]}, protoprimulagenin A on the basis of extensive NMR and MS spectral data. The saponin showed significant activity against prostate cancer cells DU145 and PC3 (EC₅₀ 1.2 and 7.4 μg/mL, respectively), while it did not affect normal cells (EC₅₀ 30 μg/mL), in contrast to the reference compound (mitoxanthrone, EC₅₀ 0.45 μg/mL). Glioblastoma cells were also significantly affected by the tested saponin (EC₅₀ 6.0 μg/mL), whereas the activity against melanoma cells was moderate (EC ₅₀ 17.5-23.2 μg/mL).

Full text of DOI:10.1016/j.carres.2013.04.005

Carbohydrate Research 375 (2013) 16–20
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
A new cytotoxic triterpene saponin from Lysimachia nummularia L.
Irma Podolak a, , Paulina Koczurkiewicz a,b, Marta Michalik b, Agnieszka Galanty a, Paweł Zajdel c,
⇑
Zbigniew Janeczko a
a Department of Pharmacognosy, Pharmaceutical Faculty, Medical College, Jagiellonian University, Medyczna 9, 30-688 Cracow, Poland
b Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Cracow, Poland
c Department of Medicinal Chemistry, Pharmaceutical Faculty, Medical College, Jagiellonian University, Medyczna 9, 30-688 Cracow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A new glycosylated triterpene 1 (named nummularoside) was isolated from the underground parts of
Received 17 February 2013
Received in revised form 2 April 2013
Accepted 4 April 2013
Lysimachia nummularia L. Its chemical structure was elucidated as 3-O-b-{{[b-
[b- -xylopyranosyl-(1?4)]-b- -glucopyranosyl-(1?4)}-[b- -glucopyranosyl-(1?2)-]-
anosyl]}, protoprimulagenin A on the basis of extensive NMR and MS spectral data. The saponin showed
significant activity against prostate cancer cells DU145 and PC3 (EC50 1.2 and 7.4 g/mL, respectively),
while it did not affect normal cells (EC50 30 g/mL), in contrast to the reference compound (mitoxan-
throne, EC50 0.45 g/mL). Glioblastoma cells were also significantly affected by the tested saponin
(EC50 6.0 g/mL), whereas the activity against melanoma cells was moderate (EC50 17.5–23.2 g/mL).
D
-xylopyranosyl-(1?2)]-
D
D
D
a-L
-arabinopyr-
Available online 12 April 2013
l
l
Keywords:
l
Lysimachia nummularia
Triterpene saponin
Cytotoxicity
l
l
Ó 2013 Elsevier Ltd. All rights reserved.
The genus Lysimachia L. comprises about 200 species, wild and
cultivated, native to temperate regions of Eurasia, and is tradition-
ally classified as one of the largest genera in Primulaceae.1 How-
ever, results from recent phylogenetic analyses suggested its re-
location to the family Myrsinaceae.2 The medicinal value of many
Lysimachia species is well known, there are reports on their use
as, for example, analgesic,3 anti-leishmanial,4 anti-helminthic5
agents and to treat cholecystitis.6 Also Lysimachia nummularia
L.—moneywort—has been used in medicine since antiquity for
indications such as diarrhoea, fever, arthritis, tuberculosis, skin dis-
eases and many other. It was listed in Codex Medicamentarius sive
Pharmacopoea Gallica (Paris, 1818).7 Extracts from L. nummularia
herb were found active against a number of microorganisms,
including Streptococcus pyogenes, Staphylococcus aureus, Salmonella
sp. and Shigella sp.8
Phytochemical studies confirmed the presence of the following
groups of compounds in this plant species: flavonoids9,10 and phe-
nolic acids11 in herb, benzoquinones12 and tannins13 in whole
plant. Genus Lysimachia, as well as other genera of the family Myr-
sinaceae (and/or Primulaceae), is characterized by the presence of
saponins with oleanane-derived sapogenols, in this, fairly rare
compounds possessing a completely saturated pentacyclic skele-
ton with 13b,28-epoxy bridge.14,15
antiproliferative potential has been attracting much interest in re-
cent years,18,19 and several compounds of the 13b,28-epoxy-olean-
ane group have exhibited such effects.20–25
In our on-going research on the cytotoxic activity of natural
products and the chemical constituents of species of Lysimachi-
a12,26,27 we investigated L. nummularia, which is one of five repre-
sentatives of this genus found on natural stands in Poland.
The present study deals with the isolation and structure
elucidation of a new triterpene glycoside 3-O-b-{{[b-
syl-(1?2)]-[b- -xylopyranosyl-(1?4)]-b- -glucopyranosyl-(1?4)}-
[b- -glucopyranosyl-(1?2)-]- -arabinopyranosyl]}, protoprimu-
D-xylopyrano-
D
D
D
a-L
lagenin A 1 (nummularoside) and evaluation of its cytotoxic
activity as well as selectivity against a panel of five human cancer
cell lines and normal cells of respective origin.
Compound 1 (Fig. 1) was isolated as a white amorphous
powder. Its molecular formula, C57H94O25, was deduced from
the HR-ESI-TOF-MS ion at m/z = 1201, 60 ([M+Na]+). This
composition was supported by 13C NMR spectral data and DEPT
135 experiments. The negative-ion FAB-MS showed fragment
ions at m/z 1045 [MꢀHꢀ132]ꢀ and 913 [MꢀHꢀ132ꢀ132]ꢀ cor-
responding to the subsequent loss of two pentose units, at m/z
1015 due to the loss of hexose unit, at m/z 751 corresponding
to the loss of
a
hexose and of two pentose units
Triterpene saponins have been reported to possess a wide range
of pharmacological activities, including anti-inflammatory, expec-
torant, vasoprotective, immunoadjuvant, antileishmanial, gastro-
protective and many other.16,17 Also, their cytotoxic and
[MꢀHꢀ162ꢀ132ꢀ132]ꢀ. This fragmentation pattern indicated a
branched sugar chain.
Acid hydrolysis on a TLC plate afforded arabinose, glucose and xylose,
which were identified by co-chromatography with authentic sugar sam-
ples. To establish their absolute configurations further experiments
were carried out, including acid hydrolysis of compound 1 (4 M HCl in
dioxane and water 1:1, v/v; 3 h, 95 °C), followed by derivatization with
⇑
Corresponding author. Tel.: +48 (12) 6205560; fax: +48 (12) 6205575.
E-mail address: mfpodola@cyf-kr.edu.pl (I. Podolak).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.04.005

I. Podolak et al. / Carbohydrate Research 375 (2013) 16–20
17
4.40 (t, J = 6.5 Hz, 1H), 4.54 (d, J = 7.6 Hz, 1H), 4.56 (d, J = 7.8 Hz,
1H), 4.70 (d, J = 7.7 Hz, 1H). These were correlated by HSQC exper-
iment to the corresponding carbon resonances at d 105.2, 105.6,
107.3, 104.6, 104.3, respectively (Table 1). In the 1H NMR spectrum
the relatively large coupling constants J1,2 of the anomeric protons
for both glucose and both xylose moieties (7.6–7.8 Hz) indicated a
b-configuration. The value of J1,2 coupling constant for the anomeric
proton of arabinose (6.5 Hz), which has been reported not to be
diagnostic on its own,36 together with NOE connectivities between
O
Glc - II
OH
O
HO
HO
Ara
OH
O
O
O
OH
O
HO
Xyl-II
O
O
O
HO
HO
HO
O
OH
OH
O
Xyl- I
HO
HO
Ara H-1, Ara H-3 and Ara H-5 indicated an a-orientation of this su-
OH
gar unit. Complete assignments of the resonances of each monosac-
charide unit were achieved by extensive NMR analyses (1H–1H
COSY, TOCSY, HSQC, HMBC, ROESY) (Table 1). The sugar sequence
of the oligosaccharide chain as well as the glycoside sites were sub-
sequently determined by HMBC spectrum. The key cross-peaks
(Fig. 2) were observed between Ara H-1 (d 4.40) and C-3 (d 91.4), be-
tween H-1 of Glc-I (d 4.70) and C-2 of Ara (d 79.5), between H-1 of
Glc-II (d 4.56) and C-4 of Ara (d 80.2), between H-1 of Xyl-I (d 4.54)
and C-2 of Glc-II (d 84.5), between H-1 of Xyl-II (d 4.34) and C-4 of
Glc-II (d 79.9). ROESY correlations were observed between dH 4.70
(Glc-I H-1) and dH 3.80 (Ara H-2), dH 4.56 (Glc-II H-1) and dH 3.89
(Ara H-4), dH 4.54 (Xyl-I H-1) and dH 3.47 (Glc-II H-2), dH 4.34
(Xyl-II H-1) and dH 3.58 (Glc-II H-4).
Glc- I
Figure 1. Structure of compound 1.
L
-cysteine methyl ester hydrochloride and o-tolyl-isothiocyanate, and
direct HPLC analysis.28 Thus, the monosaccharides were confirmed as
-arabinose, -glucose and -xylose.
L
D
D
The IR spectrum showed absorptions at 3334 (–OH), 2922 (CH)
and at 1069 (C–O–C) cmꢀ1. Of the 57 carbons in the 13C NMR spec-
trum, 30 were assigned to the triterpenoid skeleton and 27 to the
oligosaccharide moiety. Among the triterpene skeleton carbons,
two methine carbons bearing oxygen were found at d 91.4 and
77.9 ppm. The spectrum lacked signals of the olefinic carbons C-
12 and C-13 at d ꢁ122 and ꢁ145 ppm characteristic of olean-12-
enes, instead, an oxygenated quaternary carbon signal was ob-
served at d 88.4 ppm, which is the fingerprint resonance of penta-
cyclic triterpenes with a methyleneoxy bridge between C-13 and
C-17.29 This was supported by the 1H NMR spectrum in which
the C-28 protons appeared as two doublets at d 3.12 and 3.49 (each
1H, AB system, J = 7.5 Hz) characteristic of the presence of a
13b,28-epoxide.30 Moreover, the 1H NMR spectrum revealed that
1 possesses seven tertiary methyl protons at d 0.85 (3H, s, H-24),
0.91 (3H, s, H-25), 0.91 (3H, s, H-30), 0.95(3H, s, H-29), 1.05 (3H,
s, H-23), 1.14 (3H, s, H-26), 1.23 (3H, s, H-27), and the correspond-
ing methyl carbons were identified by an HSQC experiment. The
structural assignment was initiated from the long-range coupling
networks observed between the methyl protons and the adjacent
carbons from the HMBC experiment.
Correlations were observed from singlets at d 1.05 (H-23) and d
0.85 (H-24) with carbon resonances C-3 (91.4), C-4 (40.6) and C-5
(56.8); from singlet at d 0.91 (H-25) with C-1 (40.2), C-5 (56.8), C-9
(51.4), C-10 (37.8); from singlet at d 1.14 (H-26) with C-7 (35.2), C-
8 (43.3), C-9 (51.4), C-14 (45.4); from singlet at d 1.23 (H-27) with
C-8 (43.3), C-14 (45.4), C-15 (37.1), C-13 (88.4); from singlets at d
0.91 (H-30) and 0.95 (H-29) with carbons C-20 (32.4), C-19 (39.8).
Further diagnostic long-range correlation cross-peaks, which sup-
ported the presence of the 13b,28-epoxy oleanane skeleton,31,32
were observed between H-28 (d 3.12; 3.49) and C-16 (d 77.9)
and C-17(d 45.4); between H-16 (d 3.89) and C-14 (d 45.4) and
C-18 (d 52.4).
Based on the above findings, the structure of compound 1 was
elucidated as 3-O-b-{{[b-
anosyl-(1?4)]-b- -glucopyranosyl-(1?4)}-[b-
(1?2)-]- -arabinopyranosyl]}, protoprimulagenin A. This is a
D
-xylopyranosyl-(1?2)]-[b-D-xylopyr-
D
D
-glucopyranosyl-
a-L
new triterpene saponin, trivially named nummularoside.
While two glucopyranose residues attached to the 2- and 4-
positions of arabinose is a sugar chain core that was previously re-
ported in fungicidal avenacin A-1 from oat root37 and later in ca.
twenty other triterpene saponosides out of over 1500 described
in years 1996–2007, as reviewed by Dinda et al.,38 the additional
substitution of
a terminal glucose at positions 2 and 4 is
uncommon, as compared to glycosylation at C-2 and C-323,39,40 or
C-6.41,42 A five unit sugar moiety branched in such a way was iden-
tified in Anagallis arvensis (anagallosaponin I),43 however the
monosaccharides attached to the terminal glucose at positions 2
and 4 were xylose and glucose, respectively. So, to the authors’
knowledge the structure of the oligosaccharidic chain in nummul-
aroside has not been reported so far in a triterpene saponin.
Literature reports on cytotoxicity of saponins similar in struc-
ture27,34 as well as promising in vitro activity of the methanol ex-
tract from the underground parts of L. nummularia L. (50% dead
cells at 70 lg/mL against murine melanoma B16) urged us to eval-
uate in this study the properties of compound 1, isolated from this
extract, against a panel of human cancer cell lines and normal cells
of respective origin. The cytotoxicity assay was performed on glio-
blastoma astrocytoma (U375), two melanoma cell lines differing in
metastatic potential (BLM and A375), two prostate cancer cell lines
differing in metastatic potential (DU-145, PC-3) as well as on hu-
man normal skin fibroblasts (HSF) and human normal prostate cell
lines (PNT2). Compound 1 showed cytotoxic activity against all can-
cer cell lines tested (Table 2). It was most active against DU-145
cells with an EC50 value of 1.2 0.2 lg/mL. It is worth noting that
while significant activity towards both prostate cancer cell lines
of medium and high metastatic potential was seen, the compound
did not affect normal prostate cells PNT2 (EC50 = 30.0 3.2 lg/mL).
The
a-configuration of the hydroxyl group at C-16 was evident
from the 13C chemical shift in comparison to literature data (16
a
OH ca. 77 ppm; 16b OH ca. 74 ppm)29 and was confirmed by the
ROESY spectrum where cross-peaks were observed between H-16
(d 3.89) and H-15 (d 2.10), H-22 (d 1.78) and H-28 (d 3.49). The corre-
lation of Hax-3 with H-23 (d 1.05) and H-5 (d 0.73) indicated a b-con-
figuration of the hydroxyl at C-3. The glycosidic linkage at C-3 was
indicated by its downfield chemical shift (d 91.4) and from the J value
of the proton ascribable to C-3 at d 3.14 (dd, J = 11.9 and 4.6 Hz).
The above extensive NMR analysis and the comparison of 13C
data with the literature for similar compounds20,27,33–35 led us to
establish that the structure of the aglycone of compound 1 was
protoprimulagenin A.
1. Experimental
1.1. General
In the sugar region of 1H NMR spectrum, signals corresponding
to five anomeric protons were found at d 4.34 (d, J = 7.7 Hz, 1H),
Optical rotation was measured in MeOH at 20 °C on a P-2000
polarimeter. IR spectra were recorded on a Nicolet iS5 spectrometer.

18
I. Podolak et al. / Carbohydrate Research 375 (2013) 16–20
Table 1
1H and 13C NMR data (d ppm) of compound 1 in MeODa,b
Position
Aglycone
Position
Sugar moiety
dH
dC
dH (J in Hz)
dC
1
2
3
4
5
6
1.75–0.95
40.2
27.2
91.4
40.6
56.8
18.7
3-O-Ara
1.85 m–1.75
3.14 dd (11.9; 4.6)
—
0.73 d (11.1)
1.51–1.44 m
10
20
30
40
50
4.40 t (6.5)
3.80
3.80
3,89
3.54
105.6
79.5
74.4
80.2
65.7
4.20 dd (12.4; 2.8)
7
8
9
10
11
12
13
1.54 d (5.1)–1.23
—
1.26 m
—
1.63 d (12.2)–1.47
2.05–1.29
—
35.2
43.3
51.4
37.8
19.8
33.3
88.4
At C-20 Glc-I
100
200
300
400
500
600
4.70 d (7.7)
3.20
3.28
3.19
3.66
104.3
75.9
77.8
72.0
78.0
63.3
3.60
3.85
14
15
16
17
18
19
20
—
45.4
37.1
77.9
45.4
52.4
39.8
32.4
At C-40 Glc-II
2.10 dd (14.6; 5.4)–1.22
3.89
—
1.51
2.38 dd (13.9; 12.1)–1.19
—
1000
2000
3000
4000
5000
6000
4.56 d (7.9)
3.47
3.71
3.58
3.43
3.68
104.6
84.5
75.7
79.9
76.4
61.7
3.87 dd (11.3; 2.2)
21
22
23
24
25
26
2.06–1.15
1.78–1.52
1.05 s
0.85 s
0.91 s
37.4
32.1
28.4
16.7
16.7
18.8
At C-2000 Xyl-I
1IV
2IV
3IV
4IV
5IV
4.54 d (7.6)
3.25
3.38
3.52
107.3
76.0
77.6
70.9
67.4
1.14 s
3.32
3.99 dd (11.3; 5.4)
27
28
29
30
1.23 s
19.9
78.7
33.9
24.9
At C-4000 Xyl-II
3.49 d (7.5)–3.12 d (7.5)
0.95 s
0.91 s
1V
2V
3V
4V
5V
4.34 d (7.7)
3.20
3.56
3.52
105.2
74.9
77.5
70.9
67.1
3.26
3.91 dd (11.5; 5.5)
a
Assignments were confirmed by DQF-COSY, TOCSY, ROESY, HSQC and HMBC.
Overlapped signals are reported without designating multiplicity.
b
NMR spectra (1H, 13C, DEPT 135, 1H–1H DQF-COSY, ROESY, TOCSY,
HMBC, HSQC) were measured on a Bruker AVANCE II 500 MHz and
Bruker AVANCE III 600 MHz spectrometers using standard pulse se-
1.3. Isolation
The air-dried plant material (350 g) was extracted with CHCl3
(400 mL ꢂ 3) and next with MeOH with 0.5% pyridine
(400 mL ꢂ 4). The combined methanolic extract was concentrated
in vacuo, dissolved in water and eluted exhaustively with n-BuOH.
The n-BuOH-soluble fraction, after evaporation of the solvent, was
subjected to normal phase silica gel column chromatography using
CHCl3–MeOH–H2O 23:12:2. Fractions were combined on the basis
of TLC (SiO2, CHCl3–MeOH–H2O 23:12:2; sulfuric acid + heating 5–
10 min, 105 °C). Thus, six major fractions were obtained. Fraction
III (containing mainly compound of Rf 0.32) was subjected to re-
peated preparative TLC using CHCl3–MeOH–H2O (23:12:2; 8:7:1)
as solvents. Bands were removed from plates after spraying with
distilled water, the silica was extracted with MeOH and solvent
was evaporated in vacuo to yield 1 (37 mg).
quences, in CD3OD. 1H were recorded at 500.13 and 600.20 MHz, 13
C
at 125.77 and 150.94 MHz, respectively. All chemical shifts (d) are
given in ppm, and TMS was used as an internal standard. Coupling
constants are reported in Hz. Spectra were analysed using MestRec
magnetic resonance companion version 4.4 and CARA 1.8.4 (Com-
puter Aided Resonance Assignment). FAB-MS spectra were recorded
on a Finnigan MAT 95 mass spectrometer; glycerol as the matrix, Cs
ions accelerated at 13 keV. HR-ESI-TOF-MS was obtained on a
Waters SYNAPT G2-S HDMS spectrometer. Column chromatography
was carried out on Merck Kieselgel 60 (70–230 mesh). Preparative
TLC was performed on commercially precoated silica gel G plates
(Analtech, 500 l). Analytical TLC was carried out on Merck silica
gel 60 aluminium plates and the spots were visualized by spraying with
5% sulfuric acid in MeOH followed by heating for 5–10 min at 105 °C.
1.4. Identification
1.2. Plant material
3-O-b-{{[b-D-Xylopyranosyl-(1?2)]-[b-D-xylopyranosyl-(1?4)]-
The underground parts of Lysimachia nummularia L. were col-
b-
D
-glucopyranosyl-(1?4)}-[b-
D
-glucopyranosyl-(1?2)-]-a-L
-ara-
lected from natural stands in southern Poland (near Cracow) in
binopyranosyl]}, protoprimulagenin A (1, nummularoside):
2009 and were identified by dr Bozena Muszynska PhD, Depart-
White amorphous powder; mp 205 °C; ½a D20
ꢃ ꢀ3.96 (c 0.4, MeOH);
_
´
ment of Pharmaceutical Botany, Jagiellonian University, Cracow,
Poland. A voucher specimen (KFg/2009020) is deposited at the
Department of Pharmacognosy, Jagiellonian University, Cracow,
Poland.
IR (KBr); v3334 (OH), 2922 (CH), 1069 (C–O–C); HR-ESI-TOF-MS: m/
z
1201, 60 [M+Na]+; FAB-MS: m/z 1045 [MꢀHꢀ132]ꢀ, 913
[MꢀHꢀ132ꢀ132]ꢀ, 1015 [Mꢀ162]ꢀ, 751 [MꢀHꢀ162ꢀ132ꢀ132]ꢀ;
for 1H and 13C data see Table 1.

I. Podolak et al. / Carbohydrate Research 375 (2013) 16–20
19
Glc I H-1/Ara C-2
Xyl II H-1/Glc II C-4
Glc II H-1/Ara C-4
Xyl I H-1/Glc II C-2
AraH-1/C-3
4.7
4.6
4.5
4.4
F2 [ppm]
Figure 2. Detail (sugar region) of HMBC spectrum of compound 1.
Table 2
1.5.2. Determination of absolute configuration
In vitro cytotoxic activity of compound 1
Absolute configuration was determined according to a slightly
modified method, previously reported by Tanaka et al.28
Compound 1 (5 mg) was dissolved in 1.0 mL of a mixture of 4 M
HCl in dioxane and water (1:1; v/v) and was heated at 95 °C for
3 h. Then, 1.0 mL of water was added and the aglycone was ex-
tracted with ethyl acetate (3 ꢂ 3.0 mL). Subsequently, the aqueous
phase was neutralized with 2 M ammonium hydride and concen-
trated under vacuum. After drying over P2O5 for 48 h, the residue
was dissolved in anhydrous pyridine (1.0 mL) in an oven-dried
Cell line
Compound 1a (EC50
mL)
l
g/
Mitoxantroneb (EC50
mL)
lg/
Prostate panel:
PC-3
DU-145
PNT2
7.4 1.1*
1.2 0.3*
30.0 3.2
2.0 0.3*
<5.0*
0.45 0.12*
Skin panel:
A-375
BLM
23.2 1.2*
17.5 1.6*
21.3 1.8*
<5.0*
3.5 0.5*
0.2 0.04*
screw-capped vial purged with argon. Then,
ter hydrochloride (5 mg) was added and the vial was purged with
argon. The mixture was allowed to react at 60 °C for 1 h. Next, 5
of o-tolyl-isothiocyanate was added and the reaction mixture was
heated at 60 °C for an additional 1 h. Samples of authentic sugars
glucose, -glucose, -arabinose, -arabinose, -xylose and -xylose
were derivatized according to the same procedure.
L-cysteine methyl es-
HSF
Glioblastoma
U373
6.0 1.3*
<5.0*
lL
a
The EC50 value is the concentration of compound that causes 50% cell death
D
-
after 24 h of incubation. PC-3 = human prostate adenocarcinoma (high metastatic
potential); DU-145 = human prostate carcinoma (medium metastatic potential);
PNT2 = human normal prostate cells; A-375 = human malignant melanoma;
BLM = human malignant melanoma (high metastatic potential); HSF = human skin
fibroblasts; U373 = human glioblastoma astrocytoma.
L
D
L
D
L
Then, all samples were directly analysed by HPLC. The analysis
was carried out on a Dionex apparatus equipped with a PDA 100
UV–vis detector, on a 250 ꢂ 4.6 mm i.d. Hypersil GOLD C-18 (Ther-
mo EC) column at 35 °C with isocratic elution of 25% CH3CN in
50 mM H3PO4 for 40 min, at a flow rate 1 mL/min, and subsequent
washing for 15 min. The absolute configurations of the monosaccha-
b
Positive control.
Statistically significant.
*
1.5. Acid hydrolysis
rides in 1 were confirmed to be
by comparison of the retention times of monosaccharide derivatives
with those of standard sugar samples: -glucose (14.1 min), -arab-
inose (15.0 min) and -xylose (15.6 min), respectively.
D-glucose, L-arabinose and D-xylose
1.5.1. Sugar identification
Total acid hydrolysis was performed on a TLC plate with gas-
eous HCl for 25 min according to procedure described by Janeczko
et al.44 Chromatograms were developed in a mobile phase CHCl3–
MeOH–H2O 23:12:2 (twice) and visualized by spraying with ani-
line phthalate in n-BuOH followed by heating. Rf values were com-
pared with authentic sugars. Thus, the presence of Glc (Rf 0.27), Ara
(Rf 0.35) and Xyl (Rf 0.38) units was revealed.
D
L
D
1.6. Bioassay for cytotoxic activity
The human glioblastoma–astrocytoma cell line U375 was cul-
tured in Eagle’s minimum essential medium (EMEM) with 2 mM

20
I. Podolak et al. / Carbohydrate Research 375 (2013) 16–20
L
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We acknowledge the financial support of the Grant No. N405
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297636 from the Ministry of Science and Higher Education, Poland.
_
´
We wish to thank dr Bozena Muszynska for identification of the
plant material and dr Edward Szneler for recording the NMR spectra.
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Supplementary data
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Supplementary data associated with this article can be found, in
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