Triterpenoid saponins from the roots of two Gypsophila species

Article abstract of DOI:10.1016/j.phytochem.2014.02.018

Two triterpenoid saponins with two known ones have been isolated from the roots of Gypsophila arrostii var. nebulosa, and two new ones from the roots of Gypsophila bicolor. Their structures were established by extensive NMR and mass spectroscopic techniqu

Full text of DOI:10.1016/j.phytochem.2014.02.018

Phytochemistry 102 (2014) 182–188
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Triterpenoid saponins from the roots of two Gypsophila species
David Pertuit a, Sibel Avunduk b, Anne-Claire Mitaine-Offer a, Tomofumi Miyamoto c, Chiaki Tanaka c,
Thomas Paululat d, Stéphanie Delemasure e, Patrick Dutartre e, Marie-Aleth Lacaille-Dubois a,
⇑
a Laboratoire de Pharmacognosie, EA 4267, FDE/UFC, UFR Pharmacie, Université de Bourgogne, 7, Bd Jeanne d’Arc, BP 87900, 21079 Dijon Cedex, France
b Mugla University, Saglık Hizmetleri Meslek Yuksekokulu, Ulusal Egemenlik Cad. No:9 Marmaris, Mugla, Turkey
c Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
d Universität Siegen, Organische Chemie II, Naturwissenschaftlich-Technische Fakultät, Adolf-Reichwein-Str. 2, D-57076 Siegen, Germany
e Cohiro, UFR Médecine, 7, Bd Jeanne d’Arc, BP 87900, 21079 Dijon Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Two triterpenoid saponins with two known ones have been isolated from the roots of Gypsophila arrostii
var. nebulosa, and two new ones from the roots of Gypsophila bicolor. Their structures were established by
Received 20 December 2013
Received in revised form 10 February 2014
Available online 8 April 2014
extensive NMR and mass spectroscopic techniques as 3-O-b-
yl-(1?3)]-b- -glucuronopyranosylquillaic acid 28-O-b-
(1?3)]- -rhamnopyranosyl-(1?2)-[b- -glucopyranosyl-(1?4)]-b-
galactopyranosyl-(1?2)-[b- -xylopyranosyl-(1?3)]-b- -glucuronopyranosylgypsogenin 28-O-b-
yranosyl-(1?4)-[b- -glucopyranosyl-(1?3)]- -rhamnopyranosyl-(1?2)-[b- -glucopyranosyl-(1?4)]-
b- -fucopyranosyl ester (2), 3-O-b- -galactopyranosyl-(1?2)-[b- -xylopyranosyl-(1?3)]-b- -glucurono
pyranosylgypsogenin 28-O-b- -xylopyranosyl-(1?3)-b- -xylopyranosyl-(1?4)- -rhamnopyranosyl-
(1?2)-[(4-O-acetyl)-b- -quinovopyranosyl-(1?4)]-b- -fucopyranosyl ester (3), gypsogenic acid
28-O-b- -glucopyranosyl-(1?3)-{6-O-[3-hydroxy-3-methylglutaryl]-b- -glucopyranosyl-(1?6)}-b-
-galactopyranosyl ester (4). Three compounds were evaluated against one human colon cancer cell line
SW480 and one rat cardiomyoblast cell line H9c2.
D
-galactopyranosyl-(1?2)-[b-
-xylopyranosyl-(1?4)-[b- -glucopyranosyl-
-fucopyranosyl ester (1), 3-O-b-
-xylop
D-xylopyranos
D
D
D
a
-
L
D
D
D-
Keywords:
D
D
D
Gypsophila arrostii
Gypsophila bicolor
Caryophyllaceae
Triterpenoid saponins
2D NMR
D
a
-
L
D
D
D
D
D
D
D
a-L
D
D
D
D
D
Ó 2014 Elsevier Ltd. All rights reserved.
Introduction
saponins (1, 2) together with two known ones from G. arrostii
var. nebulosa, and two new triterpenoid saponins (3, 4) from G.
The genus Gypsophila (Caryophyllacae) represented by small
perennial herbs comprises more than 150 species and some of
these species have long been used as pharmaceutical and orna-
mental plants (Nie et al., 2010a). Some of them are a rich source
of saponins having a pharmaceutical and commercial importance
as medicines, detergents, adjuvants, and cosmetics (Jia et al.,
2002). A great diversity of saponins has been reported in several
species such as Gypsophila pilulifera (Arslan et al., 2012), Gypsophila
oldhamania (Luo et al., 2008), Gypsophila repens (Elbandy et al.,
2007) and Gypsophila arrostii (Frechet et al., 1991; Hostettmann
and Marston, 1995). In our continuing study on saponins from
Gypsophila genus (Elbandy et al., 2007), we have examined the
saponin fraction of the roots of G. arrostii var. nebulosa (Boiss. &
Heldr.) Bark. and Gypsophila bicolor (Freyn & Sint.) Grossh collected
in the Southwestern of Turkey. In the present paper, we report the
isolation and structure elucidation of two new triterpenoid
bicolor. The cytotoxicity of 1, 3, and 3-O-b-
(1?2)-[b- -xylopyranosyl-(1?3)]-b- -glucuronopyranosylgypsog
enin 28-O-b- -glucopyranosyl-(1?3)-[b- -xylopyranosyl-(1?4)]-
-rhamnopyranosyl-(1?2)-b- -fucopyranosyl ester was evalua
ted against a human colon cancer cell line (SW 480) and a rat car-
diomyoblast cell line (H9c2). Their structures were elucidated by
spectroscopic methods including 600 MHz 1D and 2D NMR
experiments (1H, 13C, COSY, TOCSY, NOESY, HSQC, HMBC) in com-
bination with HR-ESI-MS and by comparaison of their physical and
spectral data with literature values.
D-galactopyranosyl-
D
D
D
D
a-L
D
Results and discussion
The n-BuOH fractions obtained from the MeOH/H2O (7:3)
extract of the roots of G. arrostii var. nebulosa and G. bicolor, Ga
and Gb respectively, were fractionated by vacuum-liquid chroma-
tography (VLC) and purified by repeated medium-pressure liquid
chromatography (MPLC) on normal- and RP18 silica gel yielding
compounds 1, 2 from Ga and 3, 4 from Gb (Fig. 1). Their structures
⇑
Corresponding author. Tel.: +33 3 80 39 32 29; fax: +33 3 80 39 33 00.
E-mail address: m-a.lacaille-dubois@u-bourgogne.fr (M.-A. Lacaille-Dubois).
http://dx.doi.org/10.1016/j.phytochem.2014.02.018
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

D. Pertuit et al. / Phytochemistry 102 (2014) 182–188
30
183
29
20
19
18
21
22
12
26
11
9
17
14 16
25
10
13
1
4
O
2
8
7
28
15
R3
3
27
OR4
R1O
5
6
R2
24
23
R2
CHO
CHO
R4
S2
S2
R3
OH
H
R1
S1
S1
1
2
3
4
S1
H
H
H
CHO
COOH
S3
S4
OH
Glc1
O
GlcA
Fuc
HO
HO
O
HOOC
Xyl1
H3C
O
OH
O
HO
OH
O
S2
HO
HO
O
HO
O
O
OH
OH
Xyl2
H3C
O
O
O
O
S1
HO
HO
Rha
HO
O
OH
OH
OH
OH
Gal
O
HO
HO
Glc2
OH
H3C
O
AcO
HO
O
H3C
Fuc
CH3
OH
O
O
OH
Qui
O
O
O
Glc2
OH
S3
HO
O
HO
HO
O
H3C
O
O
OH
O
S4
HO
OH
Gal
O
OH
O
HO
Glc1
HO
HO
O
OH
OH
Xyl2
O
Rha
HO
O
OH
HO
Xyl3
OH
OH
Fig. 1. Saponins from the roots of G. arrostii var. nebulosa and G. bicolor.
3
were established mainly by spectroscopic methods including
600 MHz NMR experiments and mass spectrometry. Futhermore
two known saponins isolated from Ga were identified by
comparaison of their spectral data with literature values as 3-O-
(L
-configuration). The JH-1,H-2 values in the 1H NMR spectrum of
the glucuronic acid, xylose, galactose, glucose, fucose, quinovose
in their pyranose form (6.2–8.5 Hz) indicated their b anomeric con-
figuration and the large 1J
value of the rhamnose (168 Hz)
H-1,C-1
b-
ronopyranosylgypsogenin 28-O-b-
xylopyranosyl-(1?4)]- -rhamnopyranosyl-(1?2)-b-
nosyl ester (Nie et al., 2010b) and gypsogenin 28-O-[b-
nosyl-(1?2)-b- -galactopyranosyl-(1?3)]-[b- -glucopyranosyl-
(1?6)]-b- -galactopyranosyl ester (Elgamal et al., 1995).
Compounds 1–4 were isolated as white amorphous powders.
The monosaccharides obtained by acid hydrolysis of each com-
pound were identified by comparaison on TLC with authentic sam-
ples as glucuronic acid, xylose, galactose, glucose, fucose,
rhamnose for 1, 2, glucuronic acid, xylose, galactose, fucose, quino-
vose, rhamnose for 3 and galactose, glucose for 4. The absolute
configurations were determined by GC analysis (Hara et al.,
D
-galactopyranosyl-(1?2)-[b-
D
-xylopyranosyl-(1?3)]-b-
-glucopyranosyl-(1?3)-[b-
-fucopyra
-glucopyra
D
-glucu
confirmed that the anomeric proton was equatorial (
form).
a-pyranoid
D
D-
a
-
L
D
Compound 1 exhibited in the HR-ESI-MS a quasi-molecular ion
peak at m/z 1727.7149 [M+Na]+ (calcd. 1727.7152) compatible
with the molecular formula C76H120O42. Compound 1 showed in
ESI-MS spectrum (positive-ion mode) an ion peak at m/z 1727 indi-
cating a molecular weight of 1704. The 1H and 13C NMR spectra of 1
displayed resonances due to the triterpene part characteristic of
quillaic acid aglycon (Table 1), with six angular methyl groups at
dH 1.14 (s, H3-24), 1.00 (s, H3-25), 0.76 (s, H3-26), 1.38 (s, H3-27),
0.86 (s, H3-29) and 0.95 (s, H3-30) showing correlations in the
HSQC spectrum with their corresponding carbon at dC 10.8
(C-24), 16.4 (C-25), 17.9 (C-26), 27.3 (C-27), 33.3 (C-29) and 24.9
(C-30). Futhermore, other characteristic signals were observed
D
D
D
D
1987) to be
D for all the sugars excepted for the rhamnose

184
D. Pertuit et al. / Phytochemistry 102 (2014) 182–188
Table 1
13C NMR and 1H NMR spectroscopic data of the aglycon moieties of compounds 1–4.a,b
Position
1
2
3
4
dC
dH
dC
dH
dC
dH
dC
dH
1
2
39.3
1.06 m
1.68 m
1.76
2.04
3.86 m
–
1.32 m
0.92
37.6
0.97 m
1.56 m
1.60
1.96 m
3.68 m
–
1.26
0.83 m
1.38 m
nd
37.7
0.80
1.29
1.82
2.21
3.99
–
1.57
1.08
38.8
1.51
1.06
1.85
1.95 m
4.53
–
nd
1.88
25.6
24.1
24.6
24.2
3
4
5
6
85.9
56.4
49.1
21.3
82.7
53.9
47.3
19.7
83.9
54.9
47.3
20.5
75.2
54.0
51.2
23.8
nd
1.26
1.36
1.41
nd
7
33.7
31.8
32.3
32.0
1.60 br d (10.9)
1.53 br d (10.0)
–
1.73
–
1.75
1.90
1.41 m
–
1.61
–
1.82 m
nd
nd
–
1.61
–
1.73 br s
nd
nd
–
1.68 m
–
1.84
1.88
8
9
10
11
41.1
48.0
37.1
24.4
39.2
45.9
35.4
22.8
39.9
45.9
35.7
23.2
39.9
47.2
36.5
22.7
12
13
14
15
123.2
144.6
42.6
5.30 br t (3.6)
–
–
1.37
1.72
4.45
121.1
143.1
41.0
5.23 br s
–
–
122.8
143.9
41.8
5.31 br t
–
–
1.40
1.82
1.78
nd
123.1
143.9
41.9
5.35 br t
–
–
1.50
2.10 br t (11.6)
1.47 m
nd
36.5
26.9
nd
28.1
28.1
16
74.9
22.7
1.86 m
nd
23.6
20.8
17
18
19
49.9
42.3
48.0
–
47.5
40.6
46.6
–
47.3
41.5
45.7
–
46.7
41.3
46.2
–
2.92 dd (14.4, 4.0)
1.04 dd (13.6, 5.2)
2.28 t (13.6)
–
1.16
1.94 m
1.81 dd (14.0, 5.2)
1.89
2.82 m
1.00 m
2.23 m
–
1.28 m
nd
2.99 br d (13.6)
1.10
nd
–
1.18
nd
3.07 br d (14.3)
1.15
1.62 m
–
1.06
1.24
20
21
31.3
36.5
30.2
34.6
30.9
33.6
30.3
33.5
22
31.9
28.8
1.24 m
nd
31.9
nd
32.6
1.24
nd
23
24
25
26
27
28
29
30
211.0
10.8
16.4
17.9
27.3
177.2
33.3
24.9
9.42 s
1.14 s
1.00 s
0.76 s
1.38 s
–
209.7
10.2
15.4
16.5
25.4
174.6
32.6
24.1
9.40 s
1.09 s
0.91 s
0.68 s
1.11 s
–
210.5
10.5
15.2
17.5
26.1
176.4
32.7
23.2
9.78 s
1.34 s
0.69 s
0.90 s
1.17 s
–
181.5
12.1
15.8
17.1
25.7
175.8
32.8
23.3
–
1.50 s
0.89 s
1.02 s
1.10 s
–
0.86 s
0.95 s
0.84 s
0.90 s
0.79 s
0.74 s
0.77 s
0.79 s
a
Overlapped signals are reported without designated multiplicity; nd: not determined.
In CD3OD for 1, DMSO-d6 for 2 and pyridine-d5 for 3, 4, d in ppm, J in parentheses in Hz.
b
such as one olefinic proton at dH 5.30 (1H, br t, H-12) showing
HSQC correlation with dC 123.2 (C-12), one aldehydic proton at
dH 9.42 (s) and two oxygen bearing methine proton signals at dH
3.86 (H-3), 4.45 (H-16). The NOESY correlation between dH 0.76
(s, H3-26) and dH 1.72 (Hb-ax-15), and between dH 1.72 (Hb-ax-15)
and dH 4.45 (H-16), suggested a b-equatorial orientation of the
units, respectively. HMBC correlations between dH 4.36 (GlcA H-
1) and dC 85.9 (Agly C-3), between dH 4.60 (Xyl1 H-1) and dC 86.6
(GlcA C-3), and between dH 4.78 (Gal H-1) and dC 78.6 (GlcA C-2)
showed that the sequence linked at C-3 was identified as 3-O-
Gal-(1?2)-[Xyl-(1?3)]-GlcA, which is commonly encountered in
the genus Gypsophila (Gevrenova et al., 2006; Nie et al., 2010a; Ar-
slan et al., 2012; Elbandy et al., 2007; Frechet et al., 1991). After
subtraction of the anomeric signals of the sugars linked at the C-
3 position from the total HSQC spectrum, the signals of five sugars
linked to the aglycon through an ester linkage remained (Fuc, Rha,
Glc1, Glc2 and Xyl2). In the HSQC spectrum the deshielded ano-
meric 1H NMR signal of Fuc at dH 5.27 giving a correlation with
the shielded anomeric carbon signal at dC 95.2 suggested that the
Fuc residue was linked at C-28 through an ester linkage. This
was confirmed by an HMBC correlation between dH 5.27 (Fuc
H-1) and dC 177.2 (Agly C-28). The COSY cross peak dH 5.27 (Fuc
H-1)/dH 3.73 (Fuc H-2) and the HSQC correlation dH/dC 3.73/76.4
suggested the substitution of Fuc at the position 2. This was con-
firmed by the HMBC cross peak dH/dC 5.16/76.4 (Rha H-1/Fuc
C-2) and by the NOESY cross peak at dH/dH 5.16/3.73 (Rha H-1/
Fuc H-2). Futhermore, the HMBC correlation at dH/dC 1.26/78.6
(Rha H3-6/Rha C-4), suggested Rha C-4 to be substituted. This
was confirmed by the HMBC cross peak at dH/dC 4.67/78.6 (Xyl2
H-1/Rha C-4) and by the NOESY cross peak dH/dH 4.67/3.66 (Xyl2
H-16 and thus a
a-axial orientation of the OH group. Extensive
2D NMR analysis confirmed the structure of the aglycon to be quil-
laic acid, which is in good agreement with literature data (Guo
et al., 2000; Nord and Kenne, 1999). The chemical shift values at
dC 85.9 (C-3) and dC 177.2 (C-28), suggested that the saponin was
a bisdesmosidic glycoside with saccharide units attached to these
positions. The presence of eight sugar moieties in 1 was evidenced
by the 1H NMR spectrum which displayed eight anomeric protons
at dH 4.36 (d, J = 7.2 Hz), 4.78 (d, J = 7.6 Hz), 4.60 (d, J = 7.9 Hz), 5.27
(d, J = 7.2 Hz), 5.16 (d, J = 1.6 Hz), 4.45 (d, overlapped), 4.67 (d,
J = 7.6 Hz) and 4.53 (d, J = 7.6 Hz), giving correlations with eight
anomeric carbons at dC 104.3, 103.8, 104.7, 95.2, 101.9, 106.3,
105.1 and 105.1, respectively in the HSQC spectrum. Complete
assignments of each sugar were achieved by extensive 1D and
2D NMR analyses, allowing the identification of one b-glucurono-
pyranosyl (GlcA), one b-galactopyranosyl (Gal), two b-xylopyrano-
syl
(Xyl1,
Xyl2),
one
b-fucopyranosyl
(Fuc),
one
a-rhamnopyranosyl (Rha), two b-glucopyranosyl (Glc1, Glc2)

D. Pertuit et al. / Phytochemistry 102 (2014) 182–188
185
H-1/Rha H-4) (Gevrenova et al., 2006). At this stage, the partial se-
quence of the glycosyl ester chain at C-28 was characterized as
Xyl-(1?4)-Rha-(1?2)-Fuc-(1?28)-Agly, commonly encountered
in the genus of Gypsophila (Nie et al., 2010a; Arslan et al., 2012;
Luo et al., 2008; Elbandy et al., 2007; Frechet et al., 1991). The
two other sugar units were two terminal glucopyranosyl units
(Glc1 and Glc2) as ascertained from the analysis of 2D NMR spec-
tra. The HMBC correlation at dH/dC 1.23/83.6 (Fuc H-6/Fuc C-4),
confirmed that Fuc C-4 was substituted. The HMBCs at dH/dC
4.45/83.6 (Glc1 H-1/Fuc C-4) and 4.53/82.7 (Glc2 H-1/Rha C-3)
suggested that Glc1 was linked at Fuc C-4 and Glc2 at Rha C-3,
respectively. These linkages were confirmed by NOESY cross peaks
at dH/dH 4.45/3.74 (Glc1 H-1/Fuc H-4), and dH/dH 4.53/3.95 (Glc2
H-1/Rha H-3). This extensive 2D NMR analysis allowed the com-
plete sequencing of the glycosidic ester chain linked at C-28 as
Xyl2-(1?4)-[Glc2-(1?3)]-Rha-(1?2)-[Glc1-(1?4)]-Fuc. Thus the
(Elbandy et al., 2007). The position of the sugar unit Xyl3 was deter-
mined by observation of an HMBC correlation between dH 4.99 (Xyl3
H-1) and dC 86.5 (Xyl2 C-3) and by NOESY cross peak at dH/dH 4.99/
3.89 (Xyl3 H-1/Xyl2 H-3) proving the terminal Xyl3 to be linked at
the C-3 position of Xyl2. The position of the sugar unit Qui was
determined by observation of NOESY cross peak at dH/dH 4.93/4.04
(Qui H-1/Fuc H-4). Therefore, the complete sequence at C-28 was
characterized as Xyl3-(1?3)-Xyl2-(1?4)-Rha-(1?2)-[Qui-(1?4)]-
Fuc- which was previously characterized in Acanthophyllum pachys-
tegium (Haddad et al., 2004) and in A. glandulosum (Gaidi et al.,
2004). In the 1H NMR spectrum, the deshielded signal at dH 4.99
(Qui H-4) correlating with dC 76.1 in the HSQC spectrum suggested
the 4-position of Qui to be acetylated. This was confirmed by the
presence of a HMBC cross peak between dH 4.99 (Qui H-4) and dC
170.5. On the basis of the above results, the structure of 3 was
established as 3-O-b-
D
-galactopyranosyl-(1?2)-[b-
-glucuronopyranosylgypsogenin 28-O-b-
-xylopyranosyl-(1?4)- -L-rhamnopyranosyl-(1?
-quinovopyranosyl-(1?4)]-b- -fucopyranosyl
D
-xylopyranosyl-
(1?3)]-b-
D
D-xylopyranosyl-
structure of
(1?2)-[b- -xylopyranosyl-(1?3)]-b-
acid 28-O-b- -xylopyranosyl-(1?4)-[b-
-rhamnopyranosyl-(1?2)-[b- -glucopyranosyl-(1?4)]-b-
fucopyranosyl ester.
1
was elucidated as 3-O-b-
-glucuronopyranosylquillaic
-glucopyranosyl-(1?3)]-
D-galactopyranosyl-
(1?3)-b-
D
a
D
D
2)-[(4-O-acetyl)-b-
D
D
D
D
ester.
a-
L
D
D-
Compound 4 exhibited in the HR-ESI-MS a quasi-molecular ion
peak at m/z 1139.5254 [M+Na]+ (calcd. 1139.5250) corresponding
to the molecular formula C54H84O24. The ESI-TOF-MS spectrum
(positive-ion mode) of 4 exhibited a quasi-molecular ion peak at
m/z 1139 [M+Na]+, indicating a molecular weight of 1116. The 1H
and 13C NMR chemical shifts of the aglycon of 4 assigned from
2D NMR spectra were characteristic of gypsogenic acid (Elbandy
et al., 2007; Timité et al., 2010), with six singlet methyl signals at
dH 1.50 (H3-24), 0.89 (H3-25), 1.02 (H3-26), 1.10 (H3-27), 0.77
(H3-29) and 0.79 (H3-30) showing HSQC correlations with their
corresponding carbon at dC 12.1 (C-24), 15.8 (C-25), 17.1 (C-26),
25.7 (C-27), 32.8 (C-29), and 23.3 (C-30), respectively. Other char-
acteristic signals were observed in the HSQC spectrum such as one
olefinic proton signal at dH/dC 5.35/123.1 (H-12, br t/C-12) and one
oxygen bearing methine proton signal at dH/dC 4.53/75.2 (H-3/C-3).
The signals at dC 75.2 (C-3) and 175.8 (C-28) suggested that 4 was a
monodesmosidic saponin with a glycosyl ester linkage at C-28. The
presence of three sugar moieties in 4 was evidenced by the 1H NMR
spectrum which displayed three anomeric protons at dH 6.09 (d,
J = 8.3 Hz), 5.22 (d, J = 7.8 Hz) and 4.86 (d, J = 7.8 Hz), giving corre-
lations in the HSQC spectrum with three anomeric carbons at dC
94.5, 104.8 and 104.2 respectively. Extensive 1D and 2D NMR
anlyses allowed the identication of one Gal and two Glc (Glc1,
Glc2) respectively. The correlation in the HSQC spectrum at dH/dC
6.09/94.5 showed that the Gal residue was attached to the carbox-
ylic acid function at C-28 of the gypsogenic acid through an ester
linkage. This was confirmed by the HMBC correlation between dH
6.09 (Gal H-1)/dC/175.8 (Agly C-28). The cross peak in the HMBC
spectrum between dH 5.22 (Glc1 H-1) and dC 87.2 (Gal C-3) proved
the Glc1-(1?3)-Gal linkage. Futhermore, the HMBC correlation be-
tween dH 4.21 (Gal H-6) and dC 104.2 (Glc2 C-1) suggested a Glc2-
(1?6)-Gal linkage. This was confirmed by NOESY cross peaks at
dH/dH 4.86/4.58 (Glc2 H-1/Gal H-6), and dH/dH 5.22/4.23 (Glc1
H-1/Gal H-3). The deshielded signals of Glc2 (C-6) at dC 64.3 and
H2-6 at dH 4.53 and 4.90 observed in HSQC spectrum suggested
an acylation at this position. The presence of a dicrotalic (=3-hy-
droxy-3-methylpentanedioic acid) acyl group was ascertained by
the observation of a set of additional signals in the 1D- and 2D-
NMR spectra corresponding to a 4-carboxy-3-hydroxy-3-methyl-
1-oxobutyl moiety, which were in good agreement with literature
data (Mihci-Gaidi et al., 2010; Koike et al., 1998). This was con-
firmed by the HMBC cross peak dH/dC 4.53/171.6 Glc2 H-6/dicrota-
lic acid C-1. Thus, the structure of 4 was elucidated as gypsogenic
Compound 2 exhibited in the HR-ESI-MS a quasi-molecular ion
peak at m/z 1711.7210 [M+Na]+ (calcd. 1711.7203) corresponding
to the molecular formula C76H120O41. The ESI-MS spectrum (nega-
tive-ion mode) of 2 exhibited a quasi-molecular ion peak at m/z
1687 [MÀH]À indicating a molecular weight of 1688 followed by
a significant fragment ion peak at m/z 1527 [(MÀH)–162]À, corre-
sponding to the loss of one hexosyl moiety. Extensive 2D NMR
analysis (Table 1) showed that compounds 1 and 2 differed only
in the aglycon part at C-15 and C-16. Namely the characteristic
13C NMR signals of a methylenic and secondary alkoholic functions
in 1 at dC 36.5 (C-15) and dC 74.9 (C-16) were replaced by two
methylenic signals at dC 26.9 (C-15) and dC 22.7 (C-16) in 2, which
allowed to identify the aglycon of 2 as gypsogenin (Lacaille-Dubois
et al., 1999; Gaidi et al., 2000). Thus the structure of 2 was
elucidated as 3-O-b-
(1?3)]-b- -glucuronopyranosylgypsogenin 28-O-b-
(1?4)-[b- -glucopyranosyl-(1?3)]-
2)-[b- -glucopyranosyl-(1?4)]-b- -fucopyranosyl ester.
D-galactopyranosyl-(1?2)-[b-D-xylopyranosyl-
D
D-xylopyranosyl-
D
a-L-rhamnopyranosyl-(1?
D
D
Compound 3 exhibited in the HR-ESI-MS a quasi-molecular ion
peak at m/z 1707.7252 [M+Na]+ (calcd. 1707.7254) corresponding
to the molecular formula C77H120O40 and in the ESI-TOF-MS spec-
trum (positive-ion mode) a quasi-molecular ion peak at m/z 1707
[M+Na]+ indicating a molecular weight of 1684. The 1H and 13C
NMR data of 3 assigned by extensive 2D NMR analysis allowed
the identification of gypsogenin as aglycon (Table 1 and Table 2)
(Nie et al., 2010a,b). The deshielded chemical shift at dC 83.9
(C-3) and the shielded chemical shift at dC 176.4 (C-28) suggested
that 3 was a bisdesmosidic saponin. The presence of eight monosac-
charide moieties in 3 was evidenced by eight anomeric proton res-
onances at dH 4.64 (d, overlapped), 5.33 (d, J = 7.8 Hz), 5.18 (d,
J = 7.6 Hz), 5.82 (d, J = 8.1 Hz), 4.93 (d, J = 6.2 Hz), 6.21 (br s), 4.95
(d, J = 6.2 Hz) and 4.99 (d, J = 8.5 Hz), which were HSQC-correlated
with dC 102.9, 103.2, 103.9, 94.0, 105.7, 100.7, 106.0 and 104.9,
respectively. Extensive 2D NMR spectroscopic analysis allowed
the identification of three Xyl, one Rha, one Fuc and one quinovo-
pyranosyl (Qui) units. Additionally, the HSQC cross peak at dH/dC
2.06/20.6 and the carboxyl signal at dC 170.5 allowed the identifica-
tion of one acetyl group. Comparaison of the 2D-NMR spectra of 3
with those of 2 revealed that they share the same trisaccharide se-
quence at C-3 of the gypsogenin (Elbandy et al., 2007) and differed
only by the tetrasaccharide chain at C-28, which was identified by
2D NMR analysis as one Fuc, one Rha, one Qui and two Xyl units
(Xyl2 and Xyl3). The partial sequence characterized by HMBC corre-
lations as Xyl2-(1?4)-Rha-(1?2)-Fuc-(1?28)-Agly was already ob-
served in 1 and 2 and was in good agrement with literature data
acid 28-O-b-
D-glucopyranosyl-(1?3)-{6-O-[3-hydroxy-3-methyl-
glutaryl]-b- -glucopyranosyl-(1?6)}-b-
D
D
-galactopyranosyl ester.
Several triterpene and steroidal saponins were reported to be cyto-
toxic against a large panel of cancer cells (Lacaille-Dubois, 2005).

186
D. Pertuit et al. / Phytochemistry 102 (2014) 182–188
Table 2
13C NMR and 1H NMR spectroscopic data of the sugar moieties of compounds 1–4.a,b
1
2
3
4
dC
dH
dC
dH
dC
dH
dC
dH
3-O-
GlcA-1
2
3
4
5
6
104.3
78.6
86.6
72.0
77.7
176.1
103.8
73.6
75.2
70.7
76.4
62.6
4.36 d (7.2)
3.65
3.65
3.53 t (8.4)
3.57 d (10.0)
–
4.78 d (7.6)
3.44 m
GlcA-1
2
3
4
5
6
101.6
77.6
82.7
70.5
76.4
171.5
102.3
73.6
73.5
68.1
74.4
59.7
4.15 d (7.1)
3.40 t (8.2)
3.56 t (8.8)
3.21
3.17
–
4.54 d (7.3)
3.25
3.20
3.66
3.27
GlcA-1
2
3
4
5
6
102.9
77.5
85.2
71.3
76.7
nd
103.2
72.6
74.5
69.9
76.1
61.4
4.64
4.20
4.15
4.12
4.14
–
5.33 d (7.8)
4.25
3.98
Gal-1
Gal-1
Gal-1
2
3
4
5
6
2
3
4
5
6
2
3
4
5
6
3.42 dd (9.6, 3.2)
3.81
3.30
4.29
3.82
4.20
3.69
3.47
3.84
3.60
4.32
Xyl1-1
104.7
75.2
78.2
70.7
67.0
4.60 d (7.9)
3.22
3.30
3.50 m
3.22
Xyl1-1
102.3
73.5
76.4
69.7
65.7
4.60 d (7.6)
3.01
3.12
3.28
3.01
Xy1-1
103.9
74.4
77.3
70.2
66.4
5.18 d (7.6)
3.83
4.03
4.04
3.57
2
3
4
5
2
3
4
5
2
3
4
5
3.88 dd (11.6, 5.6)
3.69
4.19
28-O-
Fuc-1
95.2
76.4
76.5
83.6
72.0
17.0
5.27 d (7.2)
3.73
3.48
3.74
3.68 dd (8.4, 3.2)
1.23 d (6.4)
Fuc-1
93.5
73.3
76.1
82.3
69.8
16.3
5.28
Fuc-1
94.0
71.8
76.1
84.3
70.9
16.2
5.82 d (8.1)
4.46
4.16
4.04
3.94
Gal-1
94.5
72.5
87.2
68.5
76.7
68.8
6.09 d (8.3)
4.12
4.23 m
4.17 (br s)
4.02 m
4.21
2
3
4
5
6
2
3
4
5
6
3.56 t (8.8)
3.52 br d (8.8)
3.60
3.66
1.11 d (6.4)
2
3
4
5
6
2
3
4
5
6
1.44
4.58 dd (10.7, 2.0)
5.22 d (7.8)
3.98 br t (8.5)
4.10
3.96
3.90
Rha-1
101.9
71.1
82.7
78.6
69.2
18.8
5.16 d (1.6)
4.24 br t (2.7)
3.95 dd (9.2, 2.8)
3.66
3.80
1.26 d (6.0)
Rha-1
100.0
68.9
80.2
76.6
67.1
17.8
5.01 br s
4.05 m
3.81 dd (9.3, 2.8)
3.07
3.67
1.14 d (6.4)
Qui-1
105.7
74.7
74.3
76.1
70.2
17.4
4.93 d (6.2)
3.86
3.94
4.99
nd
Glc1-1
104.8
74.8
77.4
71.1
77.9
61.8
2
3
4
5
6
2
3
4
5
6
2
3
4
5
6
2
3
4
5
6
1.18
4.11
4.44 dd (10.2, 1.9)
Ac-CH3
COO
Rha-1
2
3
4
5
6
20.6
170.5
100.7
70.9
71.8
84.0
68.0
17.9
2.06 s
–
6.21 br s
4.65
4.46
4.20
Glc1-1
106.3
75.2
76.8
71.4
78.2
62.0
4.45
3.30
3.00 lt (9.2)
3.32
3.32
Glc1-1
104.9
73.3
76.6
71.1
76.7
60.8
4.30 d (7.6)
3.10
3.12
3.20
3.12
Glc2-1
104.2
74.6
77.4
71.1
74.7
64.3
4.86 d (7.8)
3.90
4.09
3.93
3.89
4.53
2
3
4
5
6
2
3
4
5
6
2
3
4
5
6
4.29
1.58 d (5.7)
3.74
3.46
nd
3.64
4.90 dd (11.2, 2.1)
Glc2-1
105.1
74.9
78.0
71.4
78.2
4.53 d (7.6)
3.29
3.32
3.30
3.28
Glc2-1
103.4
74.2
76.6
71.2
76.8
4.40 d (7.6)
3.10
3.11
3.22
3.09
Xyl2-1
106.0
74.5
86.5
68.3
66.1
4.95 d (6.2)
3.88
3.89
3.96
3.42 t (10.2)
4.10
2
3
4
5
2
3
4
5
2
3
4
5
6
62.0
3.71 m
6
60.8
3.47
nd
3.66
Xyl2-1
105.1
75.6
78.6
71.4
67.0
4.67 d (7.6)
3.10 dd (8.5, 7.6)
3.25
3.46
3.15 t -like(10.8)
3.82
Xyl2-1
104.5
73.6
76.6
69.7
65.7
4.54 d (7.6)
2.88 m
3.07
3.26
3.01
Xyl3-1
104.9
74.7
77.3
70.1
66.4
4.99 d (8.5)
3.87
4.02
4.04
3.56
2
3
4
5
2
3
4
5
2
3
4
5
3.66
4.18
Dicrotalic acid-1
2
171.6
47.0
–
2.84 m
2.86 m
–
2.72 d (15.9)
2.96 d (15.0)
–
3
4
69.9
47.2
5
6
179.5
27.2
1.49 s
a
Overlapped signals are reported without designated multiplicity; nd: not determined.
In CD3OD for 1, DMSO-d6 for 2 and pyridine-d5 for 3, 4, d in ppm, J in parentheses in Hz.
b
Therefore, the new compounds 1, 3 and the known 3-O-b-
pyranosyl-(1?2)-[b- -xylopyranosyl-(1?3)]-b- -glucuronopyrano-
sylgypsogenin 28-O-b- -glucopyranosyl-(1?3)-[b- -xylopyranosyl-(1?
4)]- -rhamnopyranosyl-(1?2)-b- -fucopyranosyl ester were
D
-galacto-
evaluated for their cytotoxic activities against one human cancer
cell line (colorectal SW480 used for the determination of antican-
cer agent activity, Finlay et al., 1986) and an embryonic rat
heart-derived cell line (cardiomyoblast H9c2 used for the study
D
D
D
D
a-L
D

D. Pertuit et al. / Phytochemistry 102 (2014) 182–188
187
of doxorubicin cytotoxicity, Wattanapitayakul et al., 2005) by XTT
assay (Jost et al., 1992). We used etoposide (topoisomerase inhib-
itor from natural origin, Orlikova et al., 2014), methotrexate (antif-
olate anticancer agent, Kodidela et al., 2014) and doxorubicin (a
DNA interacting agent with topoisomerase inhibition, Rao, 2013)
and identified by Dr. Necattin TÜRKMEN (Çukurova University,
Turkey, Science Faculty, Biology Department). The roots of G. bico-
lor (Freyn & Sint.) Grossh were collected from Van- Özalp Yeni Tul-
galı village (2100 m) in July 2002, and identified by Prof. Dr. Hasan
ÖZÇELIK (Sütçü Imam University Turkey Science Faculty, Biology
Department). The voucher specimens (N°22092013 for G. arrostii
and N° 30092013 for G. bicolor) have been deposited in the herbar-
ium of the University of Mugla-Marmaris (Turkey).
_
_
as reference drugs. The IC50 was 20
trexate on SW480 and H9c2 cells, while doxorubicin showed an
IC50 of 1 M on both cell lines. In the same experimental condi-
tions, compound 1 exhibited cytotoxicity against the two cell lines
with IC50 values of 9.97 M (SW480) and 14.67 M (H9c2). How-
lM for etoposide and metho-
l
l
l
Extraction and isolation
ever, compound 3 and the known one were inactive (IC50>20 lM).
Dried powdered roots of G. arrostii var. nebulosa (720 g) were
extracted by reflux with MeOH (2 Â 4 l). After evaporation of the
solvents under vacuum, the residue was suspended in H2O and
partitioned three times against n-BuOH (3 Â 300 ml) yielding
12 g of n-BuOH extract. An aliquot of 600 mg of this extract was
subjected to VLC over RP-18 (MeOH–H2O, 0/1, 1/1, 1/0, each
150 ml) to give three fractions (Fr.A: 128 mg, Fr.B: 109 mg, Fr.C:
300 mg). 140 mg of Fr.C was purified by MPLC (3 ml/min) over sil-
ica gel using CHCl3–MeOH–H2O (60/32/7) as eluent to give 1
(8.6 mg) and 2 (5.2 mg).
Dried powdered roots of G. bicolor (423 g) were extracted by re-
flux with MeOH (2 Â 2 l). After evaporation of the solvents under
vacuum, the residue was suspended in H2O and partitioned three
times by n-BuOH (3 Â 200 ml) yielding 18 g of n-BuOH extract.
An aliquot of this (550 mg) was subjected to VLC over RP-18, eluted
with MeOH–H2O (0/1, 1/1, 1/0, each 140 ml) to give three fractions
(Fr.D: 101 mg, Fr.E: 270 mg, Fr.F: 170 mg). 140 mg of fraction Fr.E
was fractionated by MPLC (3 ml/min) over silica gel using a CHCl3–
MeOH–H2O solvent (60/32/7) to afford twelve fractions Fr.E1–
Fr.E12. The fraction Fr.E7 (21 mg) was purified by MPLC over silica
gel using CHCl3–MeOH–H2O (60/32/7) as eluent to give 4 (3.2 mg).
The fraction Fr.F (170 mg) was fractionated by MPLC over silica gel
using a CHCl3–MeOH–H2O solvent (60/32/7) to afford seventeen
fractions Fr.F1–Fr.F17. The fractions Fr.F10–Fr.F12 (46 mg) were
purified by MPLC over silica gel in the same conditions to give 3
(5.2 mg).
Conclusion
We isolated two new triterpenoid saponins from G. arrostii var.
nebulosa roots (1, 2) and two new ones from G. bicolor roots (3, 4).
Literature survey revealed that the sequence 3-O-Gal-(1?2)-[Xyl-
(1?3)]-GlcA gypsogenin 28-O-Xyl-(1?4)-Rha-(1?2)-Fuc ester
occurs in several Gypsophila species and in some more plants of
the Caryophyllaceae family such as Acanthophyllum (Timité et al.,
2010), Psammosilene (Zhong et al., 2002), Arenaria (Gaidi et al.,
2001), and seems to represent a chemotaxonomic marker for this
family. Additionnal glycosylations and acylations contribute to
the great diversity of these saponins. Saponin 1, having quillaic
acid as aglycon showed a potential cytotoxic activity against SW
480 and H9c2 cell lines, higher than the positive controls etoposide
and methotrexate.
Experimental
General
The 1D and 2D NMR spectra (1H, 1H COSY, TOCSY, NOESY, HSQC
and HMBC) were performed using a UNITY-600 spectrometer at
the operating frequency of 600 MHz on a Varian INOVA 600 instru-
ment equipped with a Sun-4-L-X computer system (600 MHz for
1H and 150 MHz for 13C NMR spectra), for details, see (Gaidi
et al., 2000). Some spectra were recorded on a Varian VNMR-S
600 MHz spectrometer equipped with 3 mm triple resonance in-
verse and 3 mm dual broad band probeheads. All chemical shifts
(d) are given in ppm, and the samples were solubilized in CD3OD
(1), DMSO-d6 (2) pyridine-d5 (3, 4). Solvent signals were used as
internal standards. Spectra in DMSO-d6 were recorded at
T = 36 °C. The carbon type (CH3, CH2, CH, C) was determined by
DEPT experiments. ESI- (negative or positive) and ESI-MS
(positive): Q-TOF-1-Micromass spectrometer in m/z for 1, 2, and
a MicroTOF for 3, 4. The HR-ESI-MS was conducted in the posi-
tive-ion mode on a micromass Quattro LS instrument. Optical rota-
tions were taken with a Perkin-Elmer-241 polarimeter. GC analysis
was carried out on a thermoquest gas chromatograph using a
DB-1701 cap.column (30 m  0.25 mm, i.d) (J and W Scientific);
detection by FID; detector temperature, 250°, injection tempera-
ture, 230°, initial temperature was maintened at 80° for 5 min
and then raised to 270° at 15°/min; carrier gas, He. TLC and HPTLC
employed precoated Si gel plates 60 F254 (Merck). The spray re-
agent for saponins was vanilin reagent (2% mixture of conc.
H2SO4 soln. and 1% vanilin in EtOH). Isolations were carried out
using a medium-pressure liquid chromatography (MPLC) system
[Alltech pump, Büchi column (460 Â 15 mm and 230 Â 15 mm),
Büchi precolumn (110 Â 15 mm)].
Compound characterization
3-O-b-
curonopyranosylquillaic acid 28-O-b-
copyranosyl-(1?3)]- -rhamnopyranosyl-(1?2)-[b-
(1?4)]-b- -fucopyranosyl ester (1)
White, amorphous powder; [
+35 (c 0,08, MeOH); 1H NMR
D
D
-galactopyranosyl-(1?2)-[b-
D
-xylopyranosyl-(1?3)]-b-
D-glu
D
-xylopyranosyl-(1?4)-[b-
D-glu
a-L
D-glucopyranosyl-
D
25
a]
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Tables 1
and 2; ESI-MS (positive-ion mode) m/z 1727 [M+Na]+, HR-ESI-MS
(positive-ion mode) m/z 1727.7149 [M+Na]+ (calcd. for 1727.7152).
3-O-b-
glucuronopyranosylgypsogenin 28-O-b-
glucopyranosyl-(1?3)]- -rhamnopyranosyl-(1?2)-[b-
glucopyranosyl-(1?4)]-b- -fucopyranosyl ester (2)
White, amorphous powder; [
+21 (c 0.10, MeOH); 1H NMR
D
D
-galactopyranosyl-(1?2)-[b-
D
-xylopyranosyl-(1?3)]-b-
D-
D
-xylopyranosyl-(1?4)-[b-D-
a
-L
D-
D
25
a
]
(DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see Tables
1 and 2; ESI-MS (negative-ion mode) m/z 1687 [MÀH]À, HR-ESI-MS
(positive-ion mode) m/z 1711.7210 [M+Na]+ (calcd. for 1711.7203).
3-O-b-
curonopyranosylgypsogenin 28-O-b-
pyranosyl-(1?4)- -rhamnopyranosyl-(1?2)-[(4-O-acetyl)-b-
novopyranosyl-(1?4)]-b- -fucopyranosyl ester (3)
White, amorphous powder; [
(pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz), see
Tables and 2; ESI-TOF-MS (positive-ion mode) m/z 1707
D
-galactopyranosyl-(1?2)-[b-
D
-xylopyranosyl-(1?3)]-b-
-xylopyranosyl-(1?3)-b- -xylo
-qui
D-glu
D
D
a-
L
D
Plant material
D
25
a]
+7 (c 0.18 MeOH); 1H NMR
D
The roots of G. arrostii var. nebulosa (Boiss. & Heldr.) Bark. were
collected from Isparta- Islamköy (800–1200 m) in August 2002,
_
1

188
D. Pertuit et al. / Phytochemistry 102 (2014) 182–188
[M+Na]+, HR-ESI-MS (positive-ion mode) m/z 1707.7252 [M+Na]+
(calcd. for.1707.7254).
Elbandy, M., Miyamoto, T., Lacaille-Dubois, M.A., 2007. New triterpenoidal saponins
from Gypsophila repens. Helv. Chim. Acta 90, 260–270.
Elgamal, M.H.A., Soliman, H.S.M., Karawya, M.S., Mikhova, B., Duddeck, H., 1995.
Isolation of triterpene saponins from Gypsophila capillaris. Phytochemistry 38,
1481–1485.
Finlay, G.J., Wilson, W.R., Baguley, B.C., 1986. Comparison of in vitro activity of
cytotoxic drugs towards human carcinoma and leukaemia cell lines. Eur. J.
Cancer Clin. Oncol. 22, 655–662.
Frechet, D., Christ, B., Monegier, Du., Sorbier, B., Fischer, H., Vuilhorgne, M., 1991.
Four triterpenoid saponins from dried roots of Gypsophila species.
Phytochemistry 30, 927–931.
Gaidi, G., Miyamoto, T., Rustaiyan, A., Laurens, V., Lacaille-Dubois, M.A., 2000. Two
new biologically active triterpene saponins from Acanthophyllum squarrosum. J.
Nat. Prod. 63, 1497–1502.
Gaidi, G., Miyamoto, T., Lacaille-Dubois, M.A., 2001. Junceosides A–C, new triterpene
saponins from Arenaria juncea. J. Nat. Prod. 64, 1533–1537.
Gaidi, G., Miyamoto, T., Ramezani, M., Lacaille-Dubois, M.A., 2004. Glandulosides A–
D, Triterpene Saponins from Acanthophyllum glandulosum. J. Nat. Prod. 67,
1114–1118.
Gypsogenic acid 28-O-b-D-glucopyranosyl-(1?3)-{6-O-[3-hydroxy-
3-methylglutaryl]-b-
ester. (4)
D
-glucopyranosyl-(1?6)}-b-D-galactopyranosyl
25
White, amorphous powder; [
a
]
+4 (c 0.18, MeOH); 1H NMR
D
(pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz), see
Tables
1 and 2; ESI-TOF-MS (positive-ion mode) m/z 1139
[M+Na]+, HR-ESI-MS (positive-ion mode) m/z 1139.5254 [M+Na]+
(calcd. for.1139.5250).
Acid hydrolysis and GC analysis
Each compound (3 mg) was hydrolyzed with 2 N aq. CF3COOH
(5 ml) for 3 h at 95 °C. After extraction with CH2Cl2 (3 Â 5 ml),
the aq. layer was repeatly evaporated to dryness with MeOH until
neutral, and then analysed by TLC over silica gel (CHCl3–MeOH–
H2O 8/5/1) by comparaison with authentic samples. Futhermore,
the residue of sugars was dissolved in anhydrous pyridine
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(100
ll), and L-cysteine methyl ester hydrochloride (0.06 mol/l)
was added. The mixture was stirred at 60 °C for 1 h, then 150
l
l
Hara, S., Okabe, H., Mihashi, K., 1987. Gas-liquid chromatographic separation of
aldose
enantiomers
as
trimethylsilyl
ethers
of
methyl
2-
of HMDS-TMCS (hexamethyldisilazane-trimethylchlorosilane 3:1)
was added, and the mixture was stirred at 60 °C for another
30 min. The precipitate was centrifuged off, and the supernatant
was concentrated under N2 stream. The residue was partioned be-
(polyhydroxyalkyl)thiazolidine-4(R)-carboxylates. Chem. Pharm. Bull. 35,
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tween n-hexane and H2O (0.1 ml each), and the hexane layer (1 ll)
was analysed by GC (Hara et al., 1987). The absolute configurations
were determined by comparing the retention times with thiazoli-
dine derivatives prepared in a similar way from standard sugars
(Sigma-Aldrich):
cose, -fucose,
-galactose, -fucose,
D
-glucuronic acid,
-rhamnose for 1, 2;
-quinovose, -rhamnose for 3 and
D
-xylose,
-glucuronic acid,
D-galactose, D-glu-
D
L
D
D
-xylose,
-galact-
D
D
D
L
D
ose,
D-glucose for 4 were characterized by co-injection of the
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(
D
-glucuronic acid), 13.4 min (
18.6 min ( -glucose), 12.1 min (
and 11.1 min ( -quinovose).
D
-xylose), 19.6 min ( -galactose),
-fucose), 13.1 min (L-rhamnose)
D
D
D
D
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XTT cytotoxicity assay
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a-
Compounds 1, 3 and 3-O-b-
D
-galactopyranosyl-(1?2)-[b-
D
-xylo
-gl
-rhamnopyra
-fucopyranosyl ester were tested for cytotoxicity
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Med. Chem. 16, 2912–2920.
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oleanane-type glycosides from three species of Acanthophyllum. Magn. Reson.
Chem. 48, 370–374.
Wattanapitayakul, S.K., Chularojmontri, L., Herunsalee, A., Charuchongkolwongse,
S., Niumsakul, S., Bauer, J.A., 2005. Screening of antioxidants from medicinal
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pyranosyl-(1?3)]-b-
ucopyranosyl-(1?3)-[b-
nosyl-(1?2)-b-
D
-glucuronopyranosylgypsogenin 28-O-b-
D
D-xylopyranosyl-(1?4)]-
a-L
D
against one human cancer cell line SW480 (colorectal adenocarci-
noma) and one embryonic heart-derived cell line H9c2 (rat cardio-
myoblast), by means of the XTT method as described in the
literature (Jost et al., 1992). The cell lines were provided by the
Cohiro company, Dijon, France.
Acknowledgments
The authors would like to thank Dr. Memet Inan, Assoc. Prof.,
Adiyaman University, Turkey, Kahta Vocational School, 02400
Kahta/Adiyman and Prof. Dr. Saliha Kirici, Çukurova University,
Turkey, Agricultural Faculty, Department of Field Crops, 01330
Balcali, Sariçam/Adana for providing the plant material.
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