Triterpenoids in Gypsophila trichotoma Wend.

Article abstract of DOI:10.1016/j.phytol.2012.08.006

A new triterpeniod saponin 3-O-β-arabinopyranosyl-(1 → 3)-[β-galactopyranosyl-(1 → 2)]-β-glucuronopyranosyl gypsogenin (1), together with the known saponin 3-O-β-xylopyranosyl-(1 → 3)-[β-galactopyranosyl-(1 → 2)]-β-glucuronopyranosyl gypsogenin (2), and three known triterpenes gypsogenic acid (3), quillaic acid (4) and gypsogenin (5) were isolated from the roots of Gypsophila trichotoma Wend. (Caryophyllaceae). Their structures were elucidated by chemical and spectral methods. Cytotoxic activity of compounds 1 and 2 were tested against seven human cancer cell lines. Compound 1 showed cytotoxic activity against all of them, while compound 2 only against two cell lines.

Full text of DOI:10.1016/j.phytol.2012.08.006

Phytochemistry Letters 5 (2012) 752–755
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Triterpenoids in Gypsophila trichotoma Wend.
b
a
a
Maya Yotova a, Ilina Krasteva a, , Kristina Jenett-Siems , Petranka Zdraveva , Stefan Nikolov
*
a Department of Pharmacognosy, Faculty of Pharmacy, Medical University, 2 Dunav Str., 1000 Sofia, Bulgaria
b Institute of Pharmacy, Free University Berlin, Ko¨nigin-Luise-Str. 2+4, D-14195 Berlin, Germany
A R T I C L E I N F O
A B S T R A C T
Article history:
A new triterpeniod saponin 3-O-
b
-arabinopyranosyl-(1 ! 3)-[
b
-galactopyranosyl-(1 ! 2)]-
b-glucur-
Received 7 June 2012
onopyranosyl gypsogenin (1), together with the known saponin 3-O-
b
-xylopyranosyl-(1 ! 3)-[
b-
Received in revised form 2 August 2012
Accepted 17 August 2012
Available online 29 August 2012
galactopyranosyl-(1 ! 2)]-
b-glucuronopyranosyl gypsogenin (2), and three known triterpenes gypso-
genic acid (3), quillaic acid (4) and gypsogenin (5) were isolated from the roots of Gypsophila trichotoma
Wend. (Caryophyllaceae). Their structures were elucidated by chemical and spectral methods. Cytotoxic
activity of compounds 1 and 2 were tested against seven human cancer cell lines. Compound 1 showed
cytotoxic activity against all of them, while compound 2 only against two cell lines.
Keywords:
Gypsophila trichotoma
Triterpenoid saponins
Triterpenes
ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Cytotoxic activity
1. Introduction
known triterpenes of the roots of the species. The cytotoxic activity
of some of the compounds has also been tested.
The genus Gypsophila L. (Caryophyllaceae) comprises about 150
species and six of them are found in Bulgaria. Gypsophila species
are well known by their medicinal, decorative and industrial
applications. The most important classes of abundant secondary
metabolites in the genus Gypsophila are saponins including both
monodesmosidic and bidesmosidic triterpene saponins of gypso-
genin, oleanolic acid, quillaic acid, etc. Recent studies have
illustrated useful pharmacological properties of saponins, includ-
ing anticancer, immunomodulatory and cytotoxicity activities
(Bo¨ttger and Melzig, 2011). Due to their complex structures
saponins are still most efficiently produced by the plants. Since the
natural supply is limited, several research groups have explored
alternative methods for the production of these compounds such
as the in vitro cultures (Ionkov et al., 2012).
Gypsophila trichotoma Wend. is a perennial herbaceous plant,
growing in Southeast Europe, Southwest Asia, Kazakhstan, West
Mongolia, Russia and Turkmenistan. The species is spread in
Bulgaria along the Black Sea coast (Valev, 1966). Our previous
phytochemical studies resulted in the identification of triterpene
saponins, flavonoids, sterols, and volatile compounds from G.
trichotoma (Krasteva et al., 2008, 2009a,b). In this study we
describe the isolation and structure elucidation of one new
triterpenoid saponin together with one known saponin and three
2. Results and discussion
Compound 1 was isolated as a white amorphous solid from the
n-butanol extract of the roots of G. trichotoma. Its quasi-molecular
ion with m/z 939.4550 [MꢀH]ꢀ in the ESI-MS suggested a
molecular formula of C47H72O19. The 1H NMR spectrum of 1
showed signals for six tertiary methyl groups at
0.96, 1.01, 1.18 and 1.20, an olefinic proton resonance at
a broad one-proton double doublet at
and anoxygenated methine signal at
and a one proton singlet at dH = 9.46. These data suggested the
aglycone moiety of 1 to be gypsogenin (Table 1). Analysis of the
HMBC and HSQC spectra revealed an olefinic methine carbon
resonance at dC 123.2 (C-12), whereas six quaternary carbon
resonances at 56.1, 40.5, 37.1, 42.9, 47.2, and 31.06 ppm could be
assigned to C-4, C-8, C-10, C-14, C-17, and C-20, respectively. In the
d
H = 0.83, 0.93,
H = 5.26,
H = 2.87 (dd, J = 13.5, 3.8 Hz)
H = 3.88 (dd, J = 11.2, 4.5 Hz)
d
d
d
1H NMR signals for three anomeric protons appeared at
J = 7.5 Hz), H 4.86 (d, J = 7.0) and H 4.56 (d, J = 7.5) indicating three
-pyranose residues. Complete assignment of each sugar reso-
dH 4.46 (d,
d
d
b
nance including elucidation of the stereochemistry by analysis of
the coupling constants was achieved by considering the HSQC and
1H–1H COSY spectra. On this basis, the sugars were identified as
b-
glucuronic acid,
b-galactose and b-arabinose. Further evidence
was obtained by hydrolysis and co-TLC with authentic samples.
The deshielding of carbon 3 (dC 86.2) of gypsogenin indicated the
presence of the saccharidic chain in this position. This observation
was confirmed by a cross-peak in the HMBC spectrum between the
* Corresponding author. Tel.: +359 2 9236 552; fax: +359 2 9879 874.
E-mail addresses: ikrasteva@pharmfac.acad.bg, maya_jo@abv.bg,
ikrasteva@pharmfac.net (I. Krasteva).
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.phytol.2012.08.006

M. Yotova et al. / Phytochemistry Letters 5 (2012) 752–755
753
Table 1
agreement with literature data (Gevrenova et al., 2006; Guo et al.,
1998; Luo et al., 2008), compound 2 was thus identified as 3-O-
xylopyranosyl-(1 ! 3)-[ -galactopyranosyl-(1 ! 2)]- -glucuro-
NMR data compound 1 (1H 700 MHz; 13C 100.6 MHz; CD3OD).
b
-
b
b
Position
d
C, mult.
dH (J in Hz)
HMBC (H to C)
nopyranosyl gypsogenin. For the first time this substance was
isolated from Silene cucubalus Wib. (Larhsini et al., 2003).
Compounds 3–5 (Fig. 1) were identified as gypsogenic acid (3),
quillaic acid (4) and gypsogenin (5) based on their spectroscopic
data (1H and 13C NMR, HMBC, HSQC, H-H COSY) in comparison
with the literature (Gevrenova et al., 2006; Krasteva et al., 2009a;
Lavaud et al., 1998; Tori et al., 1974; Weng et al., 2008).
This is the first report on the isolation of compounds 2–5 from G.
trichotoma.
Compounds 1 and 2 were tested in a panel of human tumour
cell lines in dose dependent manner. Compound 1 showed
cytotoxicity against leukaemic cells with lymphoid (SKW-3) or
myeloid phenotype (HL-60, HL-60/Dox, K-562, NB-4, EOL-1) and
urinary bladder carcinoma cell line (CAL-29) in the highest
concentration (Table 2), while compound 2 was only active
against cell lines HL-60 and NB-4.
1
38.9, CH2
25.8, CH2
86.2, CH
56.1, qC
49.1, CH
21.2, CH2
33.2, CH2
40.5, qC
48.8, CH
37.1, qC
24.3, CH2
123.2, CH
143.7, qC
49.0, qC
28.7, CH2
23.7, CH2
47.2, qC
42.6, CH
47.2, CH2
31.6, qC
34.8, CH2
33.8, CH2
n.d.
1.71, 1.09
2
1.76, 1.98
3
3.88, dd (11.2, 4.5)
4
5
1.35
4, 10
6
1.52, 0.94
1.20, 1.53
7
8
9
1.72
5, 8, 10, 11, 25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1.93
9, 12, 13
11, 18
5.26, br. s
1.08, 1.77
2.02, 1.61
2.87, dd (13.5, 3.8)
1.13, 1.70
17
1.20, 1.41
1.54, 1.75
9.46, s
20
20
3. Experimental
3.1. General
10.6, CH3
16.1, CH3
17.7, CH3
26.3, CH3
180.7, qC
33.5, CH3
23.9, CH3
1.18, s
3, 4, 5
1.01, s
1, 9, 10
0.83, s
8, 9, 14
Melting points were measured on a Kofler hot-stage microscope
and were uncorrected. Optical rotations were measured on a
Perkin-Elmer 241 MC polarimeter. IR spectrum was obtained on a
Thermo scientific Nicolet iS10 FTIR spectrometer (Thermo
Scientific, USA). NMR (1H, HMBC, HSQC, COSY, NOESY) spectra
were recorded on a Bruker DRX 500 at 500 MHz or Bruker AVANCE
700 at 700 MHz spectrometer (Bruker, Germany) in CD3OD.
Chemical shifts were referenced to solvent peaks: dH 3.31 and
1.20, s
8, 13, 14, 15
19, 20, 21, 29
19, 20, 21, 28
0.93, s
0.96, s
GlcA
10
104.6, CH
78.4, CH
86.4, CH
71.6, CH
76.4, CH
170.8, qC
4.46, d (7.5)
3.69, m
3.70, m
3.59
3
20
30
40
d
C 49.15 for MeOH-d4. ESI-TOF was carried out on an Agilent 6210
50
3.80
ESI-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA,
USA). HPLC was run on a Shimadzu HPLC (Shimadzu, Japan) LC
10AD-liquid chromatograph binary gradient system consisting of a
pump model LC-10 AD, on-line degasser Degasys DG-1210 (VDS
Optilab), communication bus module CBM 10A, auto injector SIL-
10A, detector UV-vis SPD-10 AV, software Shimadzu CLASS LC 10,
v. 1.6. Separations were achieved on semi-prep. HPLC column Luna
60
Gal
100
200
300
400
500
600
103.9, CH
73.6, CH
75.5, CH
70.8, CH
76.8, CH
62.2, CH2
4.81, d (7.0)
3.44, t (7.2)
3.43, m
3.81, m
3.50, m
300, 400, 600
3.71, dd (5.8, 11.2), 3.80, m
5u C18 100A (250 mm ꢁ 10 mm, 5
Column chromatography (CC) was carried out with Diaion HP-
20 (Supelco, USA) and silica gel 60 and silica gel 60 C18 (40–63 m,
mm) (Phenomenex, USA).
Ara
1000
2000
3000
4000
5000
105.0, CH
72.8, CH
74.6, CH
70.0, CH
67.8, CH2
4.56, d (7.5)
30, 2000
m
3.59, t
using an over-pressure of 0.8–1.0 bar, Merck, Darmstadt,
Germany). Thin-layer chromatography (TLC) study was carried
out on pre-coated silica gel G 60 F254 plates (Merck) using solvent
systems CHCl3–MeOH (9:1), n-BuOH–AcOH–H2O (4:1:1) and
EtOAc–MeOH–AcOH–H2O (12:3:3:2). The spots were visualized
by spraying with anisaldehyde/conc. H2SO4 solution (for saponins
and sapogenins) and thymol/conc. H2SO4 for sugars followed by
heating at 110 8C. All reagents were analytical grades.
3.50, m
3.82, m
3.92, dd (1.7, 12.5), 3.60, m
3000, 4000
anomeric signal at H-10 (dH 4.46) of the glucuronic acid unit and
carbon C-3 ( C 86.2) of the aglycone. The linkages of the two other
d
monosaccharides to glucuronic acid were established from the
following HMBC correlations: Gal H-1 (dH 4.81) and GlcA C-2 (dC
3.2. Plant material
78.4), and Ara H-1 (dH 4.56) and GlcA C-3 (dC 86.4). This sequence
was confirmed by the observation of NOESY connectivities
between H-3 from the aglycone and GlcA H-1, and Gal H-1
and GlcA H-2. On the basis of these results compound 1 was
The roots of G. trichotoma Wend. (Caryophyllaceae) were
collected in August 2008 at the Black Sea coast, Bulgaria. A voucher
specimen (SO 103887) was deposited at the Herbarium of the
Faculty of Biology, Sofia University.
identified as 3-O-
b
-arabinopyranosyl-(1 ! 3)-[
b-galactopyrano-
syl-(1 ! 2)]-
b
-glucuronopyranosyl gypsogenin (Fig. 1).
Compound 2 showed a quasi-molecular ion peak at m/z
3.3. Extraction and isolation
963.4602 [M+Na]+ corresponding again to the molecular formula
47H72O19. The 1H and 13C NMR signals indicated that the
C
Air-dried powdered plant material (744 g) was exhaustively
extracted with 80% methanol (24 ꢁ 1 L). After partial evaporation
the aqueous solutions were extracted with CH2Cl2, and n-BuOH
successively. The residue from the n-BuOH layer (40 g) was
separated on a Diaion HP20 column (7.5 cm/50 cm) using a
compound had the same aglycone and a similar sugar residue as
compound 1. The anomeric proton signals appeared at dH 4.46, 4.80
and 4.57, respectively. Detailed analysis of the HSQC and 1H–1H
COSY spectra revealed that arabinose was replaced by xylose. In

754
M. Yotova et al. / Phytochemistry Letters 5 (2012) 752–755
Fig. 1. Structures of the isolated compounds.
gradient of H2O–MeOH (100:0 ! 0:100). Two hundred fractions
obtained were combined on the basis of comparable Rf values on
TLC, leading to a final 21 fractions. Frs 133–159 (9 g), rich of
saponins, were subjected to repeated flash CC on silica gel (eluted
with CH2Cl2–MeOH–H2O, 18:11:1) and RP-18 (gradient eluting
with H2O–MeOH, 100:0 ! 0:100) and further purified by semi-
3.3.2. 3-O-
-glucuronopyranosyl gypsogenin (2)
White amorphous solid; HR-ESIMS: m/z 963.4575 [M+Na]+;
calcd. for C47H72O19Na m/z 963.4565; spectral data (1H and 13C
NMR) of 2 were comparable with literature data (Guo et al., 1998;
Larhsini et al., 2003; Luo et al., 2008).
b
-xylopyranosyl-(1 ! 3)-[
b
-galactopyranosyl-(1 ! 2)]-
b
prep. HPLC using MeOH–0.03% TFA (30:70 ! 100:0, 1 mL minꢀ1
210 nm) to afford compounds 1 (7 mg) and 2 (10 mg).
,
3.3.3. (3
b,4a)-3-Hydroxyolean-12-ene-23, 28-dioic acid (gypsogenic
Fractions 87–94 (1.56 g) and 95–105 (1.50 g) were separated by
flash CC over silica gel eluting with CH2Cl2–MeOH–H2O (18:11:1)
and finally purified by semi-prep. HPLC with MeOH–0.03% TFA
acid) (3)
White crystals; HR-ESI-MS: m/z 509.3279 [M+Na]+; calcd. for
30H46O5Na m/z 509.3242. The complete structure was assigned by
C
(50:50 ! 100:0, 1 mL minꢀ1
,
210 nm) yielded compounds
3
1H and 13C NMR spectroscopic data, COSY, HSQC and HMBC
experiments help to clarify the structure, and were compared to
the literature data (Lavaud et al., 1998).
(50 mg), 4 (4 mg) and 5 (6 mg).
3.3.1. 3-O-
(1 ! 2)]- -glucuronopyranosyl gypsogenin (1)
White amorphous solid; m.p. 220–222 8C; [
b
-arabinopyranosyl-(1 ! 3)-[
b-galactopyranosyl-
b
3.3.4. (3
b,4a,16a)-3,16-Dihydroxy-23-oxoolean-12-en-28-oic acid
a
]
D
20 + 3 (c = 0.1;
(quillaic acid) (4)
MeOH); IR gmax (KBr) cmꢀ1: 2947, 2867, 1730 (C55O); HR-ESIMS
observed m/z 963.4534 [M+Na]+, 979.4325 [M+K]+, 939.4550
[MꢀH]ꢀ; calculated for 939.4667 C47H71O19. 1H and 13C NMR data
are listed in Table 1.
White amorphous solid; HR-ESI-MS: m/z 509.3258 [M+Na]+;
calcd. for C30H46O5Na 509.3243; spectral data (1H and 13C NMR) of
5 were comparable with literature data (Tori et al., 1974; Weng
et al., 2008).
3.3.5. (3b,4a)-3-Hydroxy-23-oxoolean-12-en-28-oic acid
(gypsogenin) (5)
Table 2
Citotoxicity of compounds 1 and 2.
White crystals; 5 was identified by comparison of its spectral
values with literature data (Gevrenova et al., 2006; Krasteva et al.,
2009a).
Cell line
Compound 1
IC50
g/mL)a
Compound 2
IC50
g/mL)a
(
m
(m
SKW-3
HL-60
157.0 (140.7–175.1)
112.2 (96.1–135.1)
169.0 (157.8–181.0)
157.9 (133.7–186.5)
32.3 (18.3–57.1)
>200
139.6 (127.3–153.0)
>200
3.4. Acid hydrolysis
HL-60/Dox
K-562
>200
ꢂ150
>200
>200
Compounds 1 and 2 (1 mg each) were refluxed with 7%
methanolic HCl (5 mL) for 3 h. The MeOH was evaporated, the
mixture was diluted with H2O, and the hydrolysate was parti-
tioned between EtOAc and H2O. The aqueous layer was neutralized
NB-4
EOL-1
56.5 (41.8–76.3)
CAL-29
141.7 (133.1–150.9)
a
DMSO was used as vehicle.

M. Yotova et al. / Phytochemistry Letters 5 (2012) 752–755
755
Gevrenova, R., Voutquenne-Nazabadioko, L., Harakat, D., Prost, E., Henry, M., 2006.
Complete 1H- and 13C NMR assignments of saponins from roots of Gypsophila
trichotoma Wend. Magn. Reson. Chem. 44, 686–691.
Guo, Sh., Kenne, L., Lundgren, L.N., Ro¨nnberg, B., Sundquist, B.G., 1998. Triterpenoid
saponins from Quillaja saponaria. Phytochemistry 48, 175–180.
Ionkov, T., Ionkova, I., Momekov, G., Sasheva, P., 2012. Improvement bioreactor
production of cytotoxic saponins in Astragalus membranaceus by combined
control of the bioprocesses. C. R. Acad. Bulg. Sci. 65, 1–6.
with Ag2CO3, filtered, evaporated and tested for carbohydrates by
co-TLC with authentic samples using EtOAc–MeOH–AcOH–H2O
(12:3:3:2). Each of the substances gave three spots corresponding
to GlcA, Gal for both of the compounds and Ara or Xyl respectively.
3.5. MTT dye reduction assay
Konstantinov, S.M., Eibl, H., Berger, M.R., 1999. BCR-ABL influences the antileukae-
mic efficacy of alkylphosphocholines. Br. J. Haematol. 107, 365–380.
Krasteva, I., Popov, I., Pencheva, I., Balabanova, V., Nikolov, S., 2008. Phytochemical
study of Gypsophila trichotoma Wend. (Caryophyllaceae). Quim. Nova 31,
1125–1126.
Krasteva, I., Jenet-Siems, K., Kaloga, M., Nikolov, S., 2009a. 3-O-Sulfo-triterpenoid
saponins from Gypsophila trichotoma Wend. Z. Naturforsch., B: J. Chem. Sci. 64,
319–322.
The antiproliferative/cytotoxic activity of the compounds 1 and
2 was assessed using the standard MTT-dye reduction assay
(Mosmann, 1983) with minor modifications (Konstantinov et al.,
1999) in a panel of human tumour cell lines (SKW-3, HL-60, HL-60/
Dox, K-562, NB-4, EOL-1, CAL-29).
Krasteva, I., Yotova, M., Popov, I., Zdraveva, P., Nikolov, S., 2009b. Phytochemical
study of leaves and roots of Gypsophila trichotoma Wend. using gas chroma-
tography–mass spectrometry. Pharmacia 56, 3–6.
Acknowledgements
Larhsini, M., Marston, A., Hostettman, K., 2003. Triterpenoid saponins from the roots
of Silene cucubalus. Fitoterapia 74, 237–241.
Lavaud, C., Voutquenne, L., Massiot, G., Men-Olivier, L., Das, B.C., Laprevote, O.,
Serani, L., Delaude, C., Becchi, M., 1998. Saponins from the stem bark of Filicium
decipiens. Phytochemistry 47, 441–449.
The authors would like to thank D. Yosifov, S. Konstantinov
(Laboratory for Experimental Chemotherapy, Department of
Pharmacology, Pharmacotherapy and Toxicology, Faculty of
Pharmacy, Medical University, 2 Dunav St., Sofia, Bulgaria) carrying
out the cytotoxicity testing of the compounds.
Luo, J.G., Ma, L., Kong, L.Y., 2008. New triterpenoid saponins with strong
a-
glucosidase inhibitory activity from the roots of Gypsophila oldhamiana. Bioorg.
Med. Chem. 16, 2912–2920.
Part of this research was accomplished at the Institute of
Pharmacy, Free University Berlin, Germany having the support of
DAAD Research Grant for Doctoral Candidates and Young
Academics and Scientists.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol. Methods 65,
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enes, full signal assignments including quaternary carbon signals assigned by
use of indirect 13C, 1H-spin couplings. Tetrahedron Lett. 48, 4227–4230.
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