Nebulosides A-B, novel triterpene saponins from under-ground parts of Gypsophila arrostii Guss. var. nebulosa

Article abstract of DOI:10.1016/j.bmc.2012.12.036

A bioassay-guided phytochemical analysis of the triterpene saponins from under ground parts of Gypsophila arrostii var. nebulosa allowed the isolation of two triterpene saponins; nebuloside A, B based on gypsogenin and quillaic acid aglycone. Two new oleanane type triterpenoid saponins (nebuloside A, B) and three known saponins (1-3) were isolated from the root bark of Gypsophila arrostii var. nebulosa. The structures of the two new compounds were elucidated as 3-O-β-d-galactopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]- β-d-glucuronopyranosyl quillaic acid 28-O-β-d-glucopyranosyl- (1→3)-[β-d-xylopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)] -α-l-rhamnopyranosyl-(1→2)-β-d-fucopyranosyl ester (nebuloside A) and 3-O-β-d-xylopyranosyl-(1→3)-[β-d- galactopyranosyl(1→3)-β-d-galactopyranosyl-(1→2)] -β-d-glucuronopyranosyl gypsogenin 28-O-β-d-glucopyranosyl-(1→3)- [β-d-xylopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)] -α-l-rhamnopyranosyl-(1→2)-β-d-fucopyranosyl ester (nebuloside B), on the basis of extensive spectral analysis and chemical evidence. Nebuloside A and B showed toxicity enhancing properties on saporin a type-I RIP without causing toxicity by themselves at 15 μg/mL.

Full text of DOI:10.1016/j.bmc.2012.12.036

Bioorganic & Medicinal Chemistry 21 (2013) 1279–1283
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Nebulosides A–B, novel triterpene saponins from under-ground parts
of Gypsophila arrostii Guss. var. nebulosa
b
c
Idris Arslan a, , Ali Celik , Matthias F. Melzig
⇑
a Pamukkale University, Faculty of Technology, Biomedical Engineering, TR20017 Denizli, Turkey
b Pamukkale University, Science and Arts Faculty, Biology Department, TR20017 Denizli, Turkey
c Freie Universität Berlin, Institute of Pharmacy, Königin-Luise Str. 2+4, D14195 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A bioassay-guided phytochemical analysis of the triterpene saponins from under ground parts of Gyp-
sophila arrostii var. nebulosa allowed the isolation of two triterpene saponins; nebuloside A, B based on
gypsogenin and quillaic acid aglycone. Two new oleanane type triterpenoid saponins (nebuloside A, B)
and three known saponins (1–3) were isolated from the root bark of Gypsophila arrostii var. nebulosa.
Received 29 October 2012
Revised 13 December 2012
Accepted 15 December 2012
Available online 4 January 2013
The structures of the two new compounds were elucidated as 3-O-b-
xylopyranosyl-(1?3)]-b- -glucuronopyranosyl quillaic acid 28-O-b- -glucopyranosyl-(1?3)-[b-
anosyl-(1?3)-b- -xylopyranosyl-(1?4)]- -rhamnopyranosyl-(1?2)-b- -fucopyranosyl ester (nebuloside
A) and 3-O-b- -xylopyranosyl-(1?3)-[b- -galactopyranosyl(1?3)-b- -galactopyranosyl-(1?2)]-b- -glu-
curonopyranosyl gypsogenin 28-O-b- -glucopyranosyl-(1?3)-[b- -xylopyranosyl-(1?3)-b- -xylopyrano-
syl-(1?4)]- -rhamnopyranosyl-(1?2)-b- -fucopyranosyl ester (nebuloside B), on the basis of
extensive spectral analysis and chemical evidence. Nebuloside A and B showed toxicity enhancing
properties on saporin a type-I RIP without causing toxicity by themselves at 15 g/mL.
D
-galactopyranosyl-(1?2)-[b-
D-
D
D
D-xylopyr-
Keywords:
D
a-L
D
Gypsophila arrostii Guss. var. nebulosa
Triterpene saponin
Cytotoxicity
D
D
D
D
D
D
D
a-L
D
Saporin
l
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
present a broad spectrum of biological uses, such as anti-cancer,
anti-inflammatory, ion channel blocking, immune stimulating,
antifungal, antithrombotic and hypocholesterolemic property.9–11
Gypsophila saponins are of interest in terms of their applications
in vaccines.12
Gypsophila arrostii var. nebulosa, a typical Turkish variety,
naturally found in a particular area in the Mediterranean zone of
Turkey. As a part of our ongoing investigation on bioactive triterpe-
noid saponins, we investigated the chemical constituents of G. arr-
ostii var. nebulosa which resulted in isolation of five triterpene
saponins, including two new triterpene saponins (nebuloside A
and B). We report herein the isolation and structural elucidation
of the two new saponins (nebuloside A and B) and known saponins
(one, two and three).
Saponins are a widespread class of natural compounds found in
a lot of plant species. Saponins consist of a hydrophobic triterpe-
noidal C30 or steroidal C27 backbone and one or two hydrophilic
glycoside moieties attached to the backbone. Due to this structural
composition, saponins are amphiphilic glycoconjugates which give
soap-like foams in water.1 Analysis of saponin compounds, how-
ever, can be quite compelling due to such factors as their complex-
ity, amphiphilic structure, lack of strong chromophore and prone
to form unsoluable aggregates in solution which can make
purification and quantitation difficult. Under-ground parts from
Gypsophila species are an especially rich source of triterpene sapo-
nins.2–4 They are exploited commercially for a variety of purposes
including medicines, detergents, adjuvants and cosmetics.4,5 The
most common basic structures of sapogenins isolated from Gyp-
sophila genus are mainly gypsogenin but also in fewer amounts
gypsogenic acid and quillaic acid.6 Also, saponins with an aldehyde
function at C-4 from Gypsophila pilulifera exhibited cytotoxic activ-
ity against human cancer cell line.2,7 The function of saponins in
plants is not completely elucidated but there is strong evidence
that they act as defense compounds against fungal pathogens’ at-
tack.8 In addition, many plant saponins have been isolated and
2. Results and discussion
The ESI-TOF-MS of nebuloside A, recorded in the negative mode,
gave a deprotonated molecule at m/z 1671.65 [MꢀH]ꢀ in the HR-
ESI-TOF. The molecular formula of nebuloside A was determined
as C75H118O41, with the help of NMR spectral data.
The resonances of six sp3 carbons at d 10.8, 15.4, 16.8, 25.6, 32.8,
23.3 and three sp2 carbons at d 122.1, 144.2 and 210.6 in the 13C
NMR spectrum of nebuloside A coupled with the information from
1H NMR spectrum at d 5.33, suggested nebuloside A to be a triter-
penoid glycoside with an aglycone of olean-12-ene skeleton.
⇑
Corresponding author. Tel.: +90 258 2963890; fax: +90 258 2963535.
E-mail addresses: idris.arslan@yahoo.com, iarslan@pau.edu.tr (I. Arslan).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.036

1280
I. Arslan et al. / Bioorg. Med. Chem. 21 (2013) 1279–1283
Of the 75 carbons, 30 were assigned to the triterpene moiety, 45
rhamnose units, indicated a
-orientation.14 The linkages of the su-
to the sugar units comprising three hexoses, three pentoses and
two deoxy hexoses. The 1H and 13C NMR spectra of nebuloside A
(Tables 1 and 2) displayed resonances due to the triterpene part
characteristic for quillaic acid, a common triterpene of triterpene
gar units at quillaic acid C-3 were established from the HMBC cor-
relations between the resonances of Xyl H-1 (d 5.32) and GlcA C-3
(d 86.0), Gal H-1 (d 5.58) and GlcA C-2 (d 78.4). The HMBC correla-
tion between the resonances of GlcA H-1 (d 4.88) and aglycone C-3
(d 84.7) established the attachment of the trisaccharide moiety to
C-3 position. Accordingly, the sugar arrangement in the 3-O-sac-
glycosides. The
d values of C-3 at 84.7 ppm and C-28 at
176.2 ppm, suggested that nebuloside A was a bisdesmosidic gly-
coside with saccharide units attached to these positions. The sugar
portion of nebuloside A contained in the 1H NMR spectrum, eight
anomeric proton signals at d 4.88 [d, J = 7.2 Hz, glucuronic acid
(GlcA)], 5.58 [d, J = 7.7 Hz, galactose (Gal)], 5.32 [d, J = 7.7 Hz, xy-
lose (Xyl)], 5.98 [d, J = 8.3 Hz, fucose (Fuc)], 5.79 [br s, rhamnose
(Rha)], 5.37 [d, J = 7.0 Hz, glucose (Glc)], 5.50 [d, J = 7.7 Hz, xylose
(Xyl0)], and 5.16 [d, J = 7.7 Hz, xylose (Xyl00)]. Their associated 13C
resonances assigned from HSQC and HMBC experiments were at
d 103.5, 104.0, 104.8, 94.4, 102.0, 105.2, 104.0 and 105.6, respec-
tively. On the basis of the assigned protons, the corresponding
13C resonances of the individual monosaccharides were then de-
duced from the HSQC spectrum. Further Supplementary data were
obtained from the HMBC experiment which also clarified the
assignments of some closely related protons and carbons. All the
monosaccharides were determined to be in the pyranose form
from their 13C NMR data. The anomeric centers of the glucose
caharide chain was concluded as b-
[b- -xylopyranosyl-(1?3)]-b- -glucuronopyranosyl.
The sugar linkage positions at C-28 were established from the
D-galactopyranosyl-(1?2)-
D
D
HMBC correlations between the resonances of Xyl00 H-1 (d 5.16)
and Xyl0 C-3 (d 87.8), Xyl0 H-1 (d 5.50) and Rha C-4 (d 78.4). Fur-
ther correlations between the resonances of Glc H-1 (d 5.37) and
Rha C-3 (d 82.3), Rha H-1 (d 5.79) and Fuc C-2 (d 75.9) indicated
branched sugar arrangement. The correlation between the
resonances of the aglycone C-28 (d 176.2) and Fuc H-1 (d 5.98)
provided definitive evidence for an ester linkage between the
pentasaccharide chain and the aglycone. Therefore, the sugar se-
quence at C-28 carbonyl group was determined as b-
osyl-(1?3)-[b- -xylopyranosyl-(1?3)-b- -xylopyranosyl-(1?4)]-
rhamnopyranosyl-(1?2)-b- -fucopyranosyl. Thus the structure of
nebuloside A was elucidated as 3-O-b- -galactopyranosyl-(1?2)-
[b- -xylopyranosyl-(1?3)]-b- -glucuronopyranosyl quillaic acid 28-
O-b- -glucopyranosyl-(1?3)-[b- -xylopyranosyl-(1?3)-b- -xylopyr-
-rhamnopyranosyl-(1?2)-b- -fucopyranosyl ester
D-glucopyran-
D
D
a-L-
D
D
D
D
and xylose units were each determined to have a b-configuration
D
D
D
3
based on large JH-1,H-2 values. The
a
-anomeric configurations of
anosyl-(1?4)]-
a-L
D
3
the arabinose units were deduced from their JH-1,H-2 at 7.0 Hz
(Fig. 1).
which is close to that of methyl-
and 5.0 Hz which is expected for an
a
a
-
L
-arabinopyranose (8.0 Hz)
The ESI-TOF-MS of saponin nebuloside B, recorded in the nega-
tive mode, gave a deprotonated molecule at m/z 1820.7986
[MꢀH]ꢀ in the HR-ESI-TOF. The molecular formula of nebuloside
B was determined as C81H128O45, with the help of NMR spectral
data.
-arabinose in rapid 4C1–1C4
conformational change.13 The broad singlets of the anomeric pro-
tons and the chemical shift values of C-3 and C-5 as well as the
presence of three-bond strong HMBC correlations between the res-
onances of the anomeric protons and both C-3 and C-5 of the
The resonances of six sp3 carbons at d 10.8, 16.0, 16.8, 25.6, 32.6,
23.1 and three sp2 carbons at d 122.1, 144.0 and 210.5 in the 13C
NMR spectrum of nebuloside B coupled with the information from
1H NMR spectrum at d 5.31, suggested nebuloside B to be a triter-
penoid glycoside with an aglycone of olean-12-ene skeleton.
Of the 81 carbons, 30 were assigned to the triterpene moiety,
51 to the sugar units comprising four hexoses, three pentoses
and two deoxy hexoses. The 1H and 13C NMR spectra of nebuloside
B (Tables 1 and 2) displayed resonances due to the triterpene part
characteristic for gypsogenin, a common triterpene of triterpene
glycosides. The downfield shift of C-3 (d 84.3) and C-28 (d 176.3)
of the aglycone suggested that compound nebuloside B was a bis-
desmosidic glycoside with saccharide units attached to these posi-
tions. The sugar portion of nebuloside B contained in the 1H NMR
spectrum, nine anomeric proton signals at d 4.88 [d, J = 7.2 Hz, glu-
curonic acid (GlcA)], 5.58 [d, J = 7.7 Hz, galactose (Gal)], 5.49 [d,
J = 7.7 Hz, galactose (Gal0)], 5.30 [d, J = 7.7 Hz, xylose (Xyl)], 5.98
[d, J = 8.3 Hz, fucose (Fuc)], 5.77 [br s, rhamnose (Rha)], 5.36 [d,
J = 7.0 Hz, glucose (Glc)], 5.53 [d, J = 7.7, xylose (Xyl0)], 5.14 [d,
J = 7.7 Hz, xylose (Xyl00)]. Their associated 13C resonances assigned
from HSQC and HMBC experiments were at d 103.5, 104.0, 103.0,
104.6, 94.7, 102.0, 105.2, 104.1 and 105.8, respectively. On the ba-
sis of the assigned protons, the corresponding 13C resonances of
the individual monosaccharides were then deduced from the HSQC
spectrum. Further Supplementary data were obtained from the
HMBC experiment which also clarified the assignments of some
closely related protons and carbons. All the monosaccharides were
determined to be in the pyranose form from their 13C NMR data.
The anomeric centers of the glucose, galactose and xylose units
were each determined to have a b-configuration based on large
Table 1
1H and 13C NMR spectral data for the aglycone moieties of compounds nebuloside A, B
in pyridine d5
Nebuloside A
1H (J in Hz)
Nebuloside B
1H (J in Hz)
d
d
13C
d
d
13C
1
2
3
4
5
6
7
8
1.36 m/0.78 m
1.80 m/2.08 m
4.06 m
—
1.39 m
1.14 m/1.52 m
1.46 m/1.63 m
—
38.2
25.2
84.7
55.0
48.4
20.4
32.1
40.2
47.8
36.6
23.4
1.34 m/0.79 m
1.79 m/2.05 m
4.01 m
—
1.37 m
1.12 m/1.47 m
1.46 m/1.63 m
—
37.7
25.1
84.3
55.1
48.1
20.2
32.0
40.5
47.7
36.2
23.2
9
10
11
1.65 m
—
1.78 m
1.65 m
—
1.75 m
1.85 m
1.83 m
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.33 m
—
—
122.1
144.2
41.9
27.5
23.2
47.5
41.9
46.0
30.5
33.5
32.0
210.6
10.8
15.4
16.8
25.6
176.2
32.8
23.2
5.31 m
—
—
122.1
144.0
41.7
27.6
23.2
47.5
41.7
46.0
30.0
33.5
31.7
210.5
10.8
16.0
16.8
25.6
176.3
32.6
23.1
1.38 m/2.04 m
5.16 br s
—
3.08 br d/(14 Hz)
1.18 m/1.71 m
—
1.32 m/1.18 m
1.65 m/1.96 m
9.97 s
1.45 s
0.82s
1.04 s
1.20 s
1.26 m/2.02 m
2.04 m/1.80 m
—
3.08 br d/(14 Hz)
1.18 m/1.70 m
—
1.31 m/1.15 m
1.63 m/1.98 m
9.97 s
1.47 s
0.80 s
1.03 s
1.20 s
3JH-1,H-2 values. The
a-anomeric configurations of the arabinose
units were
3
—
0.85 s
0.86 s
—
0.85 s
0.86 s
deduced from their JH-1,H-2 at 7.0 Hz which is close to that of
methyl-
a
-
L
-arabinopyranose (8.0 Hz) and 5.0 Hz which is expected
for an
a
-arabinose in rapid 4C1–1C4 conformational change.13 The

I. Arslan et al. / Bioorg. Med. Chem. 21 (2013) 1279–1283
1281
Table 2
1H and 13C NMR spectral data for the sugar moieties of compounds nebuloside A and B in pyridine d5
Nebuloside A
1H (J in Hz)
Nebuloside B
1H (J in Hz)
d
d
13C
d
d
13C
3-O-Sugars
GlcA
1
2
3
4
5
6
4.88 d (7.2)
4.36
4.26
4.48
4.50
103.5
78.4
86.0
71.5
77.0
4.88 d (7.2)
4.32
4.25
4.44
4.47
103.5
78.2
85.8
71.3
77.3
171.5
—
171.6
—
Gal
1
2
3
4
5.58 d (7.7)
4.48
4.14
4.54
3.99
104.0
73.7
75.3
70.0
76.7
61.2
5.58 d (7.7)
4.48
4.16
4.56
3.99
104.0
73.7
75.1
70.0
76.7
61.2
5
6
4.42/4.51
4.40/4.51
Gal0
1
2
3
4
5.49 d (7.7)
4.40
4.10
4.54
3.89
103.0
73.5
75.0
70.0
76.3
61.0
5
6
4.40/4.50
Xyl
1
2
3
4
5.32 d (7.7)
3.92
4.12
4.11
3.64/4.23
104.8
75.1
78.3
70.6
67.0
5.30 d (7.7)
3.92
4.10
4.11
3.64/4.23
104.6
75.1
78.3
70.6
67.0
5
28-O-sugars
Fuc
1
2
3
4
5.98 d (8.3)
4.39
4.08
3.92
3.82
94.4
75.9
75.2
72.5
72.2
16.8
5.98 d 8.3)
4.37
4.08
3.94
3.80
94.7
75.6
75.0
72.3
72.2
16.8
5
6
1.43 d (6.0)
1.43 d (6.0)
Rha
1
2
3
4
5.79 br s
5.26 br s
4.92 d (7.0)
4.52
4.42
1.65 d (6.0)
102.0
70.7
82.3
78.4
68.9
19.2
5.77 br s
5.26 br s
4.90 d (7.0)
4.52
4.42
1.65 d (6.0)
102.0
70.7
82.3
78.2
68.9
19.0
5
6
Glc
1
2
3
4
5.37 d (7.0)
4.02
4.12
3.99
3.92
105.2
75.5
78.4
72.0
78.2
62.9
5.36 d (7.0)
4.02
4.10
3.99
3.90
105.2
75.5
78.2
72.2
78.2
62.7
5
6
4.16/4.50
4.15/4.50
Xyl
1
2
3
4
5.50 d (7.7)
3.98
4.09
4.05
3.39/4.16
104.0
75.2
87.8
69.1
66.1
5.53 d (7.7)
3.96
4.10
4.05
3.40/4.16
104.1
75.0
87.7
69.1
66.2
5
Xyl0
1
2
3
4
5.16 d (7.7)
3.90
4.10
4.12
3.42/4.13
105.6
75.2
78.0
70.6
66.8
5.14 d (7.7)
3.93
4.10
4.12
3.42/4.10
105.8
75.2
78.0
70.7
66.9
5
broad singlets of the anomeric protons and the chemical shift val-
ues of C-3 and C-5 as well as the presence of three-bond strong
HMBC correlations between the resonances of the anomeric pro-
established from the HMBC correlations between the resonances of
Xyl H-1 (d 5.30) and GlcA C-3 (d 85.8), Gal0 H-1 (d 5.49) and Gal C-
3 (d 75.1), Gal H-1 (d 5.58) and GlcA C-2 (d 78.2). The HMBC cor-
relation between the resonances of GlcA H-1 (d 4.88) and aglycone
C-3 (d 84.3) established the attachment of the trisaccharide moiety
tons and both C-3 and C-5 of the rhamnose units, indicated
a-ori-
entation.14 The linkages of the sugar units at gypsogenin C-3 were

1282
I. Arslan et al. / Bioorg. Med. Chem. 21 (2013) 1279–1283
O
HO
R2
O
O
O
O
O
HO
O
HO
CHO
O
O
HO
O
O
O
O
O
OH
OR1
OH
HO
HO
HO
OH
O
HO
O
HO
OH
OH
OH
HO
HO
OR3
R1
-H
R2
-OH
-H
R3
Nebuloside A
-Xyl
-Xyl
-H
Nebuloside B
-Gal
-H
1
2
3
-OH
-H
-H
-H
-H
-H
-Xyl
Figure 1. Chemical structures of triterpene saponins from G. arrostii var. nebulosa.
140
to C-3 position. Accordingly, the sugar arrangement in the 3-O-sac-
caharide chain was concluded as b- -xylopyranosyl-(1?3)-[b-
galactopyranosyl(1?3)-b- -galactopyranosyl-(1?2)]-b- -glucurono-
D
D-
120
100
80
60
40
20
0
D
D
pyranosyl. The sugar linkage positions at C-28 were established
from the HMBC correlations between the resonances of Xyl00 H-1
(d 5.14) and Xyl0 C-3 (d 87.7), Xyl0 H-1 (d 5.53) and Rha C-4 (d
78.2). Further correlations between the resonances of Glc H-1
(d 5.36) and Rha C-3 (d 82.3), Rha H-1 (d 5.77) and Fuc C-2 (d
75.6) indicated branched sugar arrangement. The correlation be-
tween the resonances of the aglycone C-28 (d 176.3) and Fuc H-
1 (d 5.98) provided definitive evidence for an ester linkage between
the pentasaccharide chain and the aglycone. Therefore, the sugar
sequence at C-28 carbonyl group was determined as b-
anosyl-(1?3)-[b- -xylopyranosyl-(1?3)-b- -xylopyranosyl-(1?4)]-
-rhamnopyranosyl-(1?2)-b- -fucopyranosyl.
structure of nebuloside B was elucidated as 3-O-b-
syl-(1?3)-[b- -galactopyranosyl (1?3)-b-
(1?2)]-b- -glucuronopyranosyl gypsogenin 28-O-b-
syl-(1?3)-[b- -xylopyranosyl-(1?3)-b- -xylopyranosyl-(1?4)]-
rhamnopyranosyl-(1?2)-b- -fucopyranosyl ester (Fig. 1).
D-glucopyr-
Neb A
Neb B
Neb A + S
Neb B + S
S
D
D
a-L
D
Therefore
the
Figure 2. Cytotoxicity enhancing properties of isolated triterpene saponins.
D-xylopyrano-
As a result, it was concluded that G. arrostii var. nebulosa which
can be used for the combinatorial anti cancer therapy.
D
D-galactopyranosyl-
D
D-glucopyrano-
D
D
a-L-
3. Materials and methods
3.1. General methods
D
Compounds (1, 2 and 3) were determined to be Gypsophila
saponin 1, 2 and 3 by comparing its NMR data with those pub-
lished in the literature.1
The optical rotations were determined on a Perkin Elmer 192
polarimeter equipped with a sodium lamp (589 nm) and 10-cm
Saporin, a type I ribosome-inactivating protein (RIP), removes
adenine residues from the 28S ribosomal RNA as part of a process
that leads to inhibition of protein synthesis. Also, type I RIPs are
usually unable to penetrate the cellular membrane.
Isolated saponins nebuloside A and B were individually incu-
bated with or without saporin, a type-I RIPs (ribosome inactivating
proteins), to investigate the toxicity enhancing properties on
saporin.
microcell. 1H/13
C NMR spectra were recorded at 500 and
150 MHz, respectively, on a Bruker AMX-500 spectrometer. For
measuring the 1H/13C NMR analyses, the samples were prepared
by dissolving 8 mg of Gypsophila saponins in 600 mL pyridine-d5.
Chemical shifts were referred to the residual solvent signal (pyri-
dine-d5: dH 7.23, 7.58, 8.75; dC 123.8, 135.8, 150.3).
The assignments were based on DQF-COSY, TOCSY, HSQC-TOC-
SY, and HMBC experiments at 600/150 MHz. The IR spectra were
recorded with a Perkin Elmer 100 FT-IR Spectrometer. For all elec-
trospray ionization mass spectrometry (ESI-MS) analyses, an ESI-
TOF-MS instrument Agilent 6210 (Santo Clara, USA) was used in
the negative ion mode. All parameters were adjusted for producing
maximum abundance of the deprotonated molecule [MꢀH]ꢀ. HPLC
was performed with a Shimadzu LC-10AD pump and a CBM-10A
photodiode array detector with absorptions from 200 to 500 nm.
As it can clearly be seen Figure 2 that nebuloside A and B
showed significant toxicity enhancing properties on saporin a
type-I RIP without causing toxicity by themselves at 15 lg/mL
(Fig. 2) as indicated by the comparison to the toxicity of saporin
alone. In the past studies it was shown that a combination of the
cell membrane impermeable, ribosome-inactivating protein agro-
stin from Agrostemma githago L. with Saponinum album drastically
enhanced the cytotoxicity of agrostin (>10,000-fold) and saporin
(>100,000-fold).15 It seems that our results supported this study.

I. Arslan et al. / Bioorg. Med. Chem. 21 (2013) 1279–1283
1283
3.2. Plant material
was concentrated by blowing with N2. The residue was dissolved in
1-(trimethylsilyl)-imidazole and pyridine (0.1 mL), and the solu-
tion was stirred at 60 °C for 5 min. After drying the solution with
a stream of N2, the residue was partitioned between H2O and
CH2Cl2 (1 ml, 1:1 v/v). The CH2Cl2 layer was analysed by GC using
an l-Chirasil-Val column (0.32 mm ꢁ 25 m). Temperatures of the
injector and detector were 200 °C for both. A temperature gradient
system was used for the oven, starting at 100 °C for 1 min and
The underground parts of the Gypsophila arrostii Guss. var. neb-
ulosa (Boiss. & Heldr.) Barkoudah were collected from Korkuteli
district, Antalya Province of the Turkey in May 2011, and identified
by one of the authors (A. Celik). A voucher specimen of the plants is
kept (ACG1-3) at the Herbarium of the Faculty of Science and Arts,
Pamukkale University.
increasing up to 180 °C at a rate of 5 °C/min. Retention time,
cose, (14.70 min), -galactose (13.77 min), -fucose (11.20 min),
xylose (10.96 min), -rhamnose (10.71 min). From each saponin
glucose, -galactose, -fucose, -xylose,
D-glu-
3.3. Extraction and isolation
D
D
D
-
-
L
D
Roots of Gypsophila arrostii var. nebulosa L. (350 g) were crushed
and powdered. The saponins were extracted by stirring the suspen-
sion in 90% methanol (5 L) at room temperature. The MeOH solu-
tion was evaporated at 100 mbar, 40 °C. The remaining syrup
was kept at 4 °C for 24 h. The suspension was then filtered to re-
move water-insoluble tannins and other insoluble compounds.
Thereafter, the solution was fractionated (10 ꢁ 50 mL) and kept
in the freezer. In order to hydrolyse crufts like water-soluble tan-
nins each 50 mL fraction was adjusted with 30 mM sodium
hydroxide to pH ꢂ11. The chemical degradation with NaOH re-
duced the matrix complexity of the plant raw extract which facil-
itated an increased separation efficiency of the Gypsophila saponins
by HPLC. The solution was swirled 24 h at 40 °C and filtered
D
D
D
L
-rhamnose were detected.
3.7. Cytotoxicity assay
The procedure for the cytotoxic assay was performed according
to the XTT reduction method.16 In this study, the cell line ECV-304
(human endothelial cell line) was used. In brief, the ECV-304 cells
were placed in a 96-well plate at a dencity of 2000 cells per well in
100
(FBS). Saporin (6 nM) with and without nebuloside A and B
(25 g/mL) was added and cells were incubated for 72 h. For the
analysis of cytotoxicity 50 L (1 mg/mL) XTT containing 8 g/mL
lL moedified Eagles’s medium with 15% fetal bovine serum
l
l
l
phenazine methosulphate, was added to each well and cells were
incubated for 3 h at 37 °C. The absorbance of the produced forma-
zan was measured at 580 nm.
through a Nalgene™ Disposable Filterware (0.45 lm). To remove
the low molecular compounds the solution was dialyzed (MWCO
1000) against distilled water and finally freeze dried. A pre-puri-
fied saponin mixture (ꢂ300 mg) was obtained. After dissolving in
20% methanol, the saponins (0.5 mL, 40 mg) were subjected to an
Acknowledgments
UltraSep ES PHARM RP18E (7
l
m, 250 ꢁ 8 mm) column. Elution
The author thanks Stefan Böttger for skilful assistance during
purification process. This research was financially supported by
the Research Fund of the Pamukkale University. Project number
is 2009FBE017.
was performed with a gradient of methanol (A) trifluoroacetic acid
in water (0.01%) (B) starting with 20% A to 70% A over 60 min. The
flow rate was 1.5 mL/min. The separated saponins, nebuloside A
(10 mg, tR = 42.3 min), nebuloside
B
(11 mg, tR = 46.5 min),
1
(5 mg, tR = 42.3 min), (12 mg, tR = 42.3 min) and
2
3
(15 mg,
Supplementary data
tR = 42.3 min) were collected and analysed by ESI-TOF-MS. Finally
methanol was evaporated at 100 mbar, 40 °C and the saponins
were freeze dried.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.036.
3.4. Nebuloside A
References and notes
1. Weng, A.; Jenett-Siems, K.; Schmieder, P.; Bachran, D.; Bachran, C.; Görick, C.;
Thakur, M.; Fuchs, H.; Melzig, M. F. J. Chromatogr. B 2010, 878, 713.
2. Arslan, I.; Celik, A.; Chol, J. H. Fitoterapia 2012, 83, 699.
3. Hostettmann, K.; Marston, A. Saponins. In Chemistry and Pharmacology of
Natural Products; Phillipon, J. D., Ayres, D. C., Baxter, H., Eds.; Cambridge
University Press: Cambridge, 1995; pp 326–327.
4. Frechet, D.; Christ, B.; Sorbier, B. M.; Fischer, H.; Vuilhorgne, M. Phytochemistry
1991, 30, 927.
5. Tschesche, R.; Wulff, G. Fortschr. Chem. Org. Naturst. 1973, 3, 461.
6. Rochd, M.; Fons, F.; Chahlaoui, A.; Henry, M. Acta Bot. Gallica. 2004, 151, 285.
7. Bai, H.; Zhong, Y.; Xie, Y. Y.; Wang, Y. S.; Liu, L.; Zhou, L., et al Chem. Biodivers.
2007, 4, 955.
3.4.1. 3-O-b-
(1?3)]-b- -glucuronopyranosyl quillaic acid 28-O-b-
pyranosyl-(1?3)-[b- -xylopyranosyl-(1?3)-b- -xylopyranosyl-
(1?4)]- -rhamnopyranosyl-(1?2)-b- -fucopyranosyl ester
D
-Galactopyranosyl-(1?2)-[b-
D
-xylopyranosyl-
D
D
-gluco-
D
D
a
-L
D
(nebuloside A)
White amorphous powder; ½a D20
ꢃ
+0.27 (c 0.0025, distilled
water); ESI-TOF-MS (negative ion mode) [MꢀH]ꢀ at m/z
1671.6532. For 1H/13C NMR data, see Tables 1 and 2.
3.5. Nebuloside B
8. Morrissey, J. P.; Osbourn, A. E. Microbiol. Mol. Biol. Rev. 1999, 63, 708.
9. Balandrin, M. F. Commercial Utilization of Plant-Derived Saponins: An
Overview of Medicinal, Pharmaceutical and Industrial Application. In
Saponins Used in Traditional and Modern Medicine; Waller, G. R., Yamasaki, K.,
Eds.; Plenum Press: New York, 1996; pp 1–14.
10. Lacalle-Dubois, M. A.; Wagner, H. Bioactive Saponins from Plants: An Update In
Studies in Natural Products Chemistry Series; Atta-Ur-Rahman, Ed.; Elsevier
Science: Amsterdam, 2000; Vol. 21, pp 633–687.
11. Harmatha, H. Chemo-Ecological Role of Spirostanol Saponins in the Interaction
Between Plant and Insects. In Saponins in Food Feedstuffs and Medicinal Plants;
Olezsek, W., Marston, A., Eds.; Kluwer Academic Publishers: Dordrecht, The
Netherlands, 2000; pp 129–141.
3.5.1. 3-O-b-
(1?3)-b- -galactopyranosyl-(1?2)]-b-
gypsogenin 28-O-b- -glucopyranosyl-(1?3)-[b-
-xylopyranosyl-(1?4)]- -rhamno-
D
-Xylopyranosyl-(1?3)-[b-
D
-galactopyranosyl
-glucuronopyranosyl
-xylo-
D
D
D
D
pyranosyl-(1?3)-b-
pyranosyl-(1?2)-b-
D
a-L
D
-fucopyranosyl ester (nebuloside B)
White amorphous powder; ½a D20
ꢃ
+0.21 (c 0.0025, distilled
water); ESI-TOF-MS (negative ion mode) [MꢀH]ꢀ at m/z
1820.7986. For 1H/13C NMR data, see Tables 1 and 2.
12. Marciani, D. J. U.S. Patent 5,977,081; 11 Fevr 1999.
13. De Tommasi, N.; Piacente, S.; Simone, F.; Pizza, C. J. Nat. Prod. 1993, 56, 1669.
14. Jia, Z.; Koike, K.; Nikaido, T. J. Nat. Prod. 1995, 61, 1368.
15. Hebestreit, P.; Weng, A.; Bachran, C.; Fuchs, H.; Melzig, M. F. Toxicon 2006, 47,
330.
3.6. Acid hydrolysis
A solution (2 mg each) of compounds nebuloside A and B in 1 N
HCl (0.5 mL) was stirred at 80 °C for 4 h. After cooling, the solution
16. Scudiero, D.; Shoemaker, R.; Paull, K.; Monks, A.; Tierney, S.; Currens, D.; Boyd,
M. Cancer Res. 1987, 28, 4827.