Antimicrobially active hederagenin glycosides from Cephalaria elmaliensis

Article abstract of DOI:10.1055/s-0031-1298415

A phytochemical investigation of the aerial parts of Cephalaria elmaliensis resulted in the isolation of ten hederagenin-type triterpene saponins (1-10) including three new ones, elmalienoside A (1), elmalienoside B (2), elmalienoside C (3), and two known flavonoid glycosides (11-12). Their structures were identified by extensive spectroscopic techniques (1D and 2D NMR, HR ESIMS) and chemical evidence. The antimicrobial activity of the extracts and the pure compounds was evaluated by the MIC method. According to the results, all pure compounds including the new ones were found to be very active against both gram-positive and gram-negative bacteria. Georg Thieme Verlag KG Stuttgart · New York.

Full text of DOI:10.1055/s-0031-1298415

828 Original Papers
Antimicrobially Active Hederagenin Glycosides
from Cephalaria elmaliensis
Authors
Nazlı Böke Sarıkahya, Süheyla Kırmızıgül
Affiliation
Department of Chemistry, Faculty of Science, Ege University, Bornova, Izmir, Turkey
Key words
Abstract
and chemical evidence. The antimicrobial activity
"
l Cephalaria elmaliensis
!
of the extracts and the pure compounds was eval-
uated by the MIC method. According to the re-
sults, all pure compounds including the new ones
were found to be very active against both gram-
positive and gram-negative bacteria.
"
l Dipsacaceae
A phytochemical investigation of the aerial parts
of Cephalaria elmaliensis resulted in the isolation
of ten hederagenin-type triterpene saponins (1–
10) including three new ones, elmalienoside A
(1), elmalienoside B (2), elmalienoside C (3), and
two known flavonoid glycosides (11–12). Their
structures were identified by extensive spectro-
scopic techniques (1D and 2D NMR, HR ESIMS)
"
l triterpene glycoside
"
l elmalienoside A–C
"
l antimicrobial activity
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
Introduction
Preliminary TLC analysis of the MeOH extract of C.
elmaliensis suggested that it contains numerous
triterpene glycosides and a glycoside-enriched n-
BuOH fraction. Since this extract showed antimi-
crobial activity, we decided to make a phyto-
chemical examination of its BuOH fraction. As a
result, three new hederagenin type triterpene
glycosides (1–3), named elmalienoside A–C, to-
gether with nine known natural compounds, ma-
cranthoidin A (4) [18], dipsacoside B (5) [19], 3-O-
α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyra-
nosyl hederagenin 28-O-β-D-glucopyranosyl ester
(6) [20], 3-O-β-D-glucopyranosyl-(1 → 3)-α-L-
rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl
hederagenin 28-O-β-D-glucopyranosyl ester (7)
[21], α-hederin (8) [22], sapindoside B (9) [23],
macranthoside A (10) [24], tiliroside (11) [25],
and luteolin 7-O-β-D-glycoside (12) [26], were
isolated from the aerial parts of C. elmaliensis.
The structures of these compounds were identi-
fied by extensive spectroscopic analysis, including
1D, 2D NMR, HR ESIMS data, and chemical evi-
dence. The antimicrobial activity of the extracts
and the pure isolated compounds was evaluated
by the MIC method.
!
Triterpene glycosides are constituents of many
plant drugs and folk medicines, especially from
the Orient. Consequently, great interest has been
shown in their characterization and in the inves-
tigation of their pharmacological and biological
properties. The list of biological activities associ-
ated with triterpene glycosides is very long. Cer-
tain attributes of saponins, such as their fungici-
dal and piscicidal effects, have been known for
many years, while new activities are continually
being discovered [1–3]. In light of all of this
knowledge, we decided to investigate the glyco-
sidic compounds in Cephalaria elmaliensis (Dipsa-
caceae). Previous phytochemical investigations
on different Cephalaria species have reported a
number of triterpene saponins, iridoids, flavo-
noids, alkaloids, lignans, and their glycosidic com-
pounds [4–15]. Triterpene saponins are the major
examples of these types of substances [4–11].
Furthermore, many Cephalaria species have been
used in traditional medicine for many years due
to their antimicrobial, antifungal, antioxidant, al-
leviative, anti-infectant, hypothermic, relaxant,
insecticidal, and cytotoxic activities [7–13,16].
Cephalaria elmaliensis (Hub.-Mor. & Matthews) is
an endemic species which is widespread mainly
in the southwestern Anatolia region [17].
received
revised
accepted
July 15, 2011
February 28, 2012
March 8, 2012
Bibliography
DOI http://dx.doi.org/
10.1055/s-0031-1298415
Published online April 11, 2012
Planta Med 2012; 78: 828–833
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Prof. Dr. Suheyla Kirmizigul
Department of Chemistry
Faculty of Science
Ege University
35100 Bornova, Izmir
Turkey
Phone: + 902323112816
Fax: + 902323888264
suheyla.kirmizigul@ege.edu.tr
Sarıkahya NB and Kırmızıgül S. Antimicrobially Active Hederagenin… Planta Med 2012; 78: 828–833

Original Papers 829
Materials and Methods
!
Table 1 1H‑NMR data for compounds 1, 2, and 3a–c
.
General
Position
1
2
3
Optical rotations and FTIR spectra were recorded on a Rudolph
Research Analytical Autopol I automatic polarimeter and on an
ATI Mattson Genesis Series Fourier transform infrared spectro-
photometer, respectively. High resolution electrospray ionization
mass spectra (HR ESIMS) and the 1D and 2D NMR spectra were
recorded on a Bruker LC micro-Q‑TOF instrument and on a Varian
AS 400 MHz spectrometer in DMSO-d6 with TMS as an internal
standard, respectively. Medium pressure liquid chromatography
(MPLC) was carried out using a Buchi system (Buchi C-605
pumps, coupled to a UV detector) with Buchi glass columns (15/
460, 36/460). Lichroprep RP-18 (25–40 µm; Merck) and silica gel
60 (0.063–0.200 mm; Merck) were used both for column chro-
matography and MPLC studies. Thin-layer chromatography (TLC)
was performed on F254 (Merck) and RP-18 F254 s (Merck) pre-
coated aluminum sheets. They were detected after developing
with suitable solvent systems spraying with 20% H2SO4 solution
followed by heating at 120°C for 3 minutes.
3
5
3.46, m
3.48, m
1.15, m
1.47, m
5.13, brs
3.10, 3.23, m
0.54, s
3.48, m
1.15, m
1.47, m
5.13, brs
nd, 3.38, m
0.54, s
1.18, m
9
1.48, m
12
23
24
25
26
27
29
30
5.14, brs
3.10, 3.34, m
0.55, s
0.85, s
0.84, s
0.84, s
0.66, s
0.65, s
0.64, s
1.07, s
1.05, s
1.06, s
0.85, s
0.84, s
0.84, s
0.85, s
0.84, s
0.84, s
Ara at C-3
4.30, d (6.0)
3.47, m
Ara at C-3
4.31, d (5.6)
3.50, m
3.48, m
3.56, m
3.28, 3.66, m
Rha
Ara at C-3
4.32, d (6.0)
3.50, m
3.47, m
3.57, m
3.31, 3.69, m
Rha
1
2
3
4
5
3.48, m
3.54, m
3.35, 3.66, m
Rha
1
2
3
4
5
6
5.10, brs
3.91, m
5.04, brs
3.68, m
3.44, m
3.15, m
3.66, m
1.05, d (6.0)
5.13, brs
3.78, m
3.60, m
3.16, m
3.80, m
1.05, d (6.0)
Xyl
Plant material
3.63, m
Cephalaria elmaliensis (Hub.-Mor. & Matthews) was collected
from Antalya-Elmalı, Çiğlikara, at about 1750 m altitude in July
2007. It was identified by H. Sümbül and R.S. Göktürk (Depart-
ment of Biology, Faculty of Art and Science, Akdeniz University).
A voucher specimen has been deposited (No: R.S. Göktürk 6097)
at the Herbarium Research and Application Centre of the Akdeniz
University.
3.37, m
3.80, m
1.07, d (6.4)
Glc
1
2
3
4
5
6
4.29, d (7.2)
3.04, m
4.29, d (7.2)
3.06, m
3.07, m
3.21, m
3.00, 3.68, m
3.31, m
3.11, m
3.08, m
Extraction and isolation
3.42, 3.62, m
Glc I at C-28
5.20, d (8.0)
3.10, m
The air-dried and powdered aerial parts of the plant (1.35 kg)
were extracted with MeOH (3 × 2 L) overnight at room tempera-
ture, and the combined extracts were concentrated under vacuum
at ~ 40°C. The MeOH residue was extracted with n-BuOH‑H2O,
1:1 (3 × 300 mL) solvent system, and then the n-BuOH fraction
was defatted with hexane (10 × 50 mL) to remove chlorophyll
and oily substances. The repurified n-BuOH extract which was
found to be the most biologically active fraction, was taken under
investigation. A part of the n-BuOH fraction (42.5 g) was chroma-
tographed on a RP silica gel via vacuum liquid chromatography
(VLC) eluted with an MeOH‑H2O solvent system (0:100 →
100:0% with increasing polarity by an MeOH 10% gradient) to
give 11 fractions. The combined fractions 7 and 8 were loaded in-
to a silica gel column (1200 g, 115 × 6.0 cm) with solvent systems
CHCl3-MeOH‑H2O (80:20:2; 70:30:3; 61:32:7) and MeOH (3 L
each) to give 30 fractions. The 24th of these fractions (1.2 g) was
purified on a silica gel column (110 g, 85 × 2.0 cm) eluted with
CHCl3-MeOH‑H2O (80:20:2) to give the first novel compound, 1
(111 mg), and the known compound 4 (109 mg). Compound 2
(1.3 g) was obtained by silica gel column (185 g, 135 × 2.0 cm)
chromatography eluted with the CHCl3-MeOH‑H2O (90:10:1)
solvent system from the combined fractions 15 and 17 as a sec-
ond novel natural product. Fraction 18 (2 g) was purified by silica
gel CC (185 g, 135 × 2.0 cm) using solvent system CHCl3-
MeOH‑H2O (80:20:2) to afford the last new saponin, compound
3 (210 mg), and a known compound, 5 (349 mg). Fraction 10
(538 mg) was re-chromatographed on a silica gel column (55 g,
120 × 1.35 cm) using solvent system CHCl3-MeOH‑H2O (90:
10:1) to yield compound 6 (195 mg). The combined fractions 12
and 13 (435 mg) were further separated by a silica gel column
(40 g, 100 × 1.2 cm) chromatography eluting with the CHCl3-
Glc at C-28
5.20, d (8.0)
3.10, m
Glc at C-28
5.20, d (6.8)
3.10, m
1
2
3
4
5
6
3.11, m
3.30, m
3.21, m
3.22, m
3.20, m
3.29, m
3.22, m
3.21, m
3.10, m
3.45, 3.91, m
Gal
3.66, 3.91, m
Gal
3.56, 3.90, m
Gal
1
2
3
4
5
6
4.44, d (7.2)
3.80, m
4.44, d (7.6)
3.80, m
4.44, d (7.2)
3.80, m
3.10, m
3.10, m
3.11, m
3.26, m
3.26, m
3.26, m
3.42, m
3.41, m
3.43, m
3.40, 3.60, m
3.39, 3.59, m
3.43, 3.60, m
a 1H‑NMR data (δ) were measured in DMSO-d6 at 400 MHz. b Coupling constants (J) in
Hz are given in parentheses. c nd: Not detected
MeOH‑H2O (90:10:1) solvent system to give compound 7
(83 mg). The 9th of the VLC fractions was subjected to MPLC [sil-
ica gel, 36 × 460 mm Buchi glass column, solvent systems CHCl3-
MeOH‑H2O 90:10:1; 80:20:2; 70:30:3; 61:32:7 (900 mL
each), flow rate: 30 mL/min, max. pressure: 30 bar], and 14 frac-
tions were obtained. Compound 8 (200 mg) was purified by silica
gel column (35 g, 45 × 1.8 cm) chromatography with the solvent
system CHCl3-MeOH‑H2O (90:10:1) from the 6th of these frac-
tions. Fraction 7 was loaded into a silica gel column (40 g,
100 × 1.2 cm) with the solvent system CHCl3-MeOH‑H2O
(90:10:1) to give compounds 9 (69 mg) and 10 (89 mg). Com-
pound 11 (42.3 mg) was obtained from the 2nd-4th combined
fractions of the thirty fractions obtained earlier by MPLC (silica
gel, 15 × 460 mm Buchi glass column, solvent system CHCl3-
Sarıkahya NB and Kırmızıgül S. Antimicrobially Active Hederagenin… Planta Med 2012; 78: 828–833

830 Original Papers
Table 2 13C‑NMR data for compounds 1, 2, and 3a–c
.
1
2
3
1
2
3
Position
Aglycon
Position
Sugars
Ara at C-3
103.9
74.6
Ara at C-3
103.6
74.9
Ara at C-3
103.8
74.1
73.9
68.9
65.7
Rha
1
2
39.0
26.1
80.1
43.0
46.9
17.8
32.5
39.7
47.8
36.6
23.7
122.4
144.2
42.0
27.9
23.2
46.7
41.4
46.3
31.0
33.9
32.3
63.1
13.8
16.3
17.4
26.2
176.0
33.5
24.1
39.0
39.0
26.0
80.1
43.0
46.9
17.8
32.0
nd
1
2
3
4
5
26.0
80.0
42.9
46.9
17.8
32.5
39.6
47.8
36.6
23.6
122.4
144.2
42.0
27.9
23.2
46.6
41.4
46.2
30.9
33.9
32.3
63.2
13.7
16.3
17.4
26.2
176.0
33.5
24.1
3
73.9
73.5
4
68.8
68.5
5
65.6
65.0
6
Rha
Rha
7
1
2
3
4
5
6
100.5
70.0
100.6
71.0
100.2
70.7
81.6
71.6
68.5
18.4
Xyl
8
9
47.9
36.6
23.6
82.3
71.2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
71.5
72.7
68.6
68.8
122.4
144.2
42.0
27.9
23.2
46.6
41.4
46.3
30.9
34.0
32.3
63.2
13.8
16.3
17.4
26.2
176.0
33.5
24.0
18.5
18.4
Glc
1
2
3
4
5
6
105.4
74.6
106.3
74.4
77.4
69.8
66.4
77.0
70.5
77.4
61.6
Glc I at C-28
94.8
Glc at C-28
94.8
Glc at C-28
94.8
1
2
3
4
5
6
71.3
71.3
71.3
77.0
77.0
76.9
69.8
69.8
70.1
77.1
77.1
77.0
68.8
68.8
68.7
Gal
Gal
Gal
1
2
3
4
5
6
101.8
71.9
101.8
71.9
101.7
71.8
73.0
73.0
72.9
68.1
68.1
68.0
75.1
75.0
74.9
62.0
62.0
62.0
a 13C‑NMR data (δ) were measured in DMSO-d6 at 100 MHz. b The assignments are based on DEPT, COSY, NOESY, HMQC, and HMBC experiments. c nd: Not determined
MeOH 9:1, flow rate: 10 mL/min, max. pressure: 50 bar). The last
compound 12 (40 mg) was purified from the 3rd fraction of VLC
firstly by MPLC [silica gel, 36 × 460 mm Buchi glass column, sol-
vent system CHCl3-MeOH‑H2O 90:10:1; 80:20:2; 70:30:3;
61:32:7 (1 L each), flow rate: 30 mL/min, max. pressure:
30 bar], and secondly by MeOH-acetone (1:3) precipitation pro-
cedures.
positive-ion HR ESIMS m/z 1229.5948 [M + Na]+ (calcd. for
C58H94O26Na, 1229.5926).
Alkaline hydrolysis
Pure compounds 1–3 (20 mg each) were refluxed with 5% KOH in
water solution (5 mL) at 80°C for 2 h. The reaction mixtures were
neutralized with 5% HCl in water solution and then concentrated
to dryness. The residues were extracted with n-BuOH‑H2O (1:1),
and all of the organic layers were evaporated to dryness to afford
prosapogenins [7]. Finally, they were analyzed by 1H NMR and HR
ESIMS methods. Alkaline hydrolysis of 1, 2, and 3 afforded three
known triterpene glycosides which were also isolated in this
study, namely, macranthoside A (10) [24], α-hederin (8) [22],
and sapindoside B (9) [23], respectively.
Elmalienoside A (1): white, amorphous powder (111 mg); [α]D25
–
7.4 (c 2.4, MeOH); IR (KBr) νmax 3368, 2941, 1741, 1591, 1388,
1261, 1055 cm−1
(DMSO-d6, 100 MHz), see l Tables 1 and 2, respectively; posi-
tive-ion HR ESIMS m/z 1259.6054 [M
;
1H NMR (DMSO-d6, 400 MHz) and 13C NMR
"
+
Na]+ (calcd. for
C
59H96O27Na, 1259.6031).
Elmalienoside B (2): white, amorphous powder (1.3 g); [α]2D5–8.6
(c 3.1, MeOH); IR (KBr) νmax 3399, 2944, 1752, 1640, 1458, 1389,
1261, 1059 cm−1
(DMSO-d6, 100 MHz), see l Tables 1 and 2, respectively; posi-
;
1H NMR (DMSO-d6, 400 MHz) and 13C NMR
Acid hydrolysis
"
The identification of the monosaccharide units of the glycosides
was performed by the microhydrolysis technique on a TLC plate
and GC‑MS analysis with authentic samples [7,27,28]. Each com-
pound (5 mg) was hydrolyzed with 1 N HCl (2 mL) for 6 h at 90°C.
After extraction with CHCl3 (3 × 5 mL), the aqueous layer was
evaporated to dryness and then analyzed by TLC over silica gel
(CHCl3-MeOH‑H2O-gAcOH/16:9:2:2) by comparison with au-
tive-ion HR ESIMS m/z 1097.5487 [M
53H86O22Na, 1097.5503).
Elmalienoside C (3): white, amorphous powder (210 mg); [α]D25
+
Na]+ (calcd. for
C
–
11.0 (c 2.0, MeOH); IR (KBr) νmax 3368, 2942, 1736, 1595, 1459,
1388, 1261, 1055 cm−1
NMR (DMSO-d6, 100 MHz), see l Tables 1 and 2, respectively;
;
1H NMR (DMSO-d6, 400 MHz) and 13C
"
Sarıkahya NB and Kırmızıgül S. Antimicrobially Active Hederagenin… Planta Med 2012; 78: 828–833

Original Papers 831
thentic monosaccharide samples. Furthermore, the residue of
monosaccharides was dissolved in anhydrous pyridine (1 mL);
then 1 mL of HMDS-TMCS (hexamethyldisilazane−trimethyl-
chlorosilane, 1:1) was added, and the mixture was stirred at
70°C for 1 h. The mixture was concentrated under an N2 stream
and dissolved in n-hexane for GC‑MS analyses. A mixture which
contained standard monosaccharide units was silylated by the
same procedure, too. L-Arabinose, L-rhamnose, D-xylose, D-galac-
tose, and D-glucose were detected by co-injection of the hydroly-
sate with standard silylated monosaccharide samples for GC‑MS
analyses. The retention times of the standard monosaccharides L-
arabinose, L-rhamnose, D-xylose, D-galactose, and D-glucose were
found to be 14.77, 15.25, 18.37, 29.22, and 30.70 min, respective-
ly. The monosaccharide moieties of the samples were determined
as L-arabinose, L-rhamnose, D-galactose, and D-glucose giving
peaks at 14.74, 15.18, 29.20, and 30.66 min for 1, 14.76, 15.15,
29.22, and 30.64 min for 2, 14.75, 15.16, 29.20, and 30.68 min for
3, respectively. D-xylose was found only for 3, giving a peak at
18.26 min.
Antimicrobial assay
In vitro antibacterial activity tests of all compounds were eval-
uated using minimum inhibitory concentration (MIC) measure-
ments against eight bacterial strains [four gram-negative: Esche-
richia coli (ATCC 23999), Pseudomonas aeruginosa (ATCC 27853),
Salmonella typhimurium (CCM 5445), and Klebsiella pneumoniae
(CCM 2318); and four gram-positive: Staphylococcus aureus
(ATCC 6538-P), Staphylococcus epidermidis (ATCC 12228), Bacillus
cereus (ATCC 7064), and Enterococcus faecalis (ATCC 29212)]. The
bacterial strains were inoculated on Mueller-Hinton broth (Difco)
and incubated for 24 h at 37 0.1°C. The inocula from 24-h broth
cultures were adjusted to 0.5 McFarland standards. The dilution
series of the compounds were prepared in test tubes then trans-
ferred to the broth in 96-well microtiter plates. Final concentra-
tions were from 256 to 0.5 µg/mL in the medium. The last well
containing 100 µL of nutrient broth without compounds and
10 µL of the inocula on each strip was used as a negative control.
All plates were covered with a sterile plate sealer and incubated
at 37°C for 24 h. The MIC is defined as the lowest concentration
that appeared clear against a black background. Samples from
clear wells were subcultured by plotting onto Mueller Hinton
agar. Gentamycin (Sigma) was used as the positive control. Dilu-
tions were prepared from 128 to 0.25 µg/mL concentrations in
microtiter plates [29].
Fig. 1 Chemical structures of elmalienoside A–C (1–3) and the seven
known triterpene glycosides (4–10).
glucopyranosyl ester (7) [21]. The monodesmosidic ones were
elucidated as α-hederin (8) [22], sapindoside B (9) [23], and ma-
cranthoside A (10) [24]. The other two known compounds, 11
and 12, were determined as tiliroside [25] and luteolin-7-O-β-D-
glycoside [26], respectively, by comparison of their physical and
spectroscopic data with those reported in the literature. Tiliro-
side (11) and compounds 6, 7, and 10 were isolated for the first
time from the Cephalaria genus. All known compounds were
identified by comparison of the results of the 1D, 2D NMR, and
HRESIMS techniques and chemical methods with reference data.
Supporting information
The 1D, 2D NMR, and HR ESIMS spectra of the novel compounds
1–3 (elmalienoside A–C) from C. elmaliensis are available as Sup-
porting Information.
Results and Discussion
!
Twelve compounds were isolated from the n-BuOH fraction of
Cephalaria elmaliensis. Seven compounds (4–10) (l Fig. 1) were
"
"
Compound 1 (elmalienoside A) (l Fig. 1) was isolated as an
determined to be known hederagenin-type triterpene glycosides
which can be classified as monodesmosidic and bisdesmosidic
ones. The bisdesmosidic glycosides were macranthoidin A (4)
[18], dipsacoside B (5) [19], 3-O-α-L-rhamnopyranosyl-(1 → 2)-
α-L-arabinopyranosyl hederagenin 28-O-β-D-glucopyranosyl es-
ter (6) [20], and 3-O-β-D-glucopyranosyl-(1 → 3)-α-L-rhamno-
pyranosyl-(1 → 2)-α-L-arabinopyranosyl hederagenin 28-O-β-D-
amorphous solid and had a molecular formula C59H96O27, deter-
mined by HR ESIMS (1259.6054 [M + Na]+) and confirmed by 1H
13
"
and C NMR data (see l Tables 1 and 2). The IR spectrum of 1
suggested a glycoside (3368, 1055 cm−1) and indicated the pres-
ence of a carbonyl group (1741 cm−1) in the molecule. The 1H
NMR spectrum of 1 showed characteristic singlets due to six qua-
ternary methyl groups at δH 1.07, 0.85 (× 3), 0.66, and 0.55, a hy-
Sarıkahya NB and Kırmızıgül S. Antimicrobially Active Hederagenin… Planta Med 2012; 78: 828–833

832 Original Papers
Table 3 MIC results (µg/mL) of compounds 1–12 from C. elmaliensis.
Compounds
Microorganisms
1
2
3
4
5
6
7
8
9
10
11
12
Gentamycin
S. aureus
32
16
16
16
8
32
16
16
16
8
64
32
32
32
32
32
4
64
32
32
32
16
64
4
32
16
32
16
16
32
2
32
32
16
16
16
32
4
32
16
16
16
8
32
16
16
16
16
16
2
32
32
16
16
16
32
1
32
32
16
16
8
8
16
16
16
8
32
16
16
16
8
1.0
1.0
S. epidermidis
S. typhimurium
E. coli
1.0
1.0
B. cereus
4.0
K. pneumoniae
E. faecalis
32
2
32
2
32
1
32
8
16
2
16
4
4.0
16.0
2.0
P. aeruginosa
16
16
32
32
16
16
16
16
16
16
16
16
droxymethyl group at δH 3.46 (m), and an olefinic proton at
H 5.14 (brs). The 13C NMR spectrum also revealed the signals for
(C-3 of aglycone), δH 5.20 (Glc) and δC 176.0 (C-28 of aglycone)
confirmed the linkage points of the sugar moieties to the agly-
cone. A correlation between δH 5.04 and δC 74.9 revealed the
(1 → 2) linkage between rhamnose and arabinose. Moreover, a
characteristic correlation between δH 4.44 and δC 68.8 revealed
the (1 → 6) linkage between galactose and glucose. On the basis
of the above results, the structure of 2 was elucidated as 3-O-α-
L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl hederagenin
28-O-β-D-galactopyranosyl-(1 → 6)-β-D-glucopyranosyl ester which
was named elmalienoside B.
δ
six quaternary carbons at δC 33.5, 26.2, 24.1, 17.4, 16.3, and 13.8;
an oxygen-bearing methine carbon at δC 80.1, a hydroxymethyl
group at δC 63.1, a set of olefinic carbons at δC 144.2 and 122.4,
and one carbonyl carbon at δC 176.0 confirmed that 1 has a hed-
eragenin aglycone [30]. The C-3 oxymethine carbon and C-28
carbonyl carbon were observed at δC 80.1 and 176.0, respectively,
which suggested that 1 is a 3,28-bisdesmoside of hederagenin. In
addition to these signals, the 1H and 13C NMR spectra of 1 con-
tained five clear signals for anomeric protons and carbons at δH
5.20 (d, J = 8.0 Hz), 5.10 (brs), 4.44 (d, J = 7.2 Hz), 4.30 (d,
J = 6.0 Hz), 4.29 (d, J = 7.2 Hz), and δC 105.4, 103.9, 101.8, 100.5,
94.8. All proton signals for the sugar moieties were associated
with COSY, NOESY, and HMQC spectra. The acid hydrolysis of
compound 1 gave L-arabinose, L-rhamnose, D-glucose, D-galac-
tose, and hederagenin whereas alkaline hydrolysis gave com-
pound 10 (macranthoside A). The identityofeachmonosaccharide
was confirmed by TLC and GC‑MS analyses comparing them
with authentic samples [27,28]. These results were also con-
firmed by HMBC data. In the HMBC spectrum, the H-3 proton
of the aglycone at δH 3.46 and the H-1 proton of glucose I at δH
5.20 showed long-range correlations with C-1 of the arabinose
moiety at δC 103.9 and C-28 of the aglycone at δC 176.0, respec-
tively. On the other hand, long-range correlations between the H-
1 proton of D-glucose at δH 4.29 and the C-3 carbon of L-rhamnose
at δC 82.3, the H-1 proton of L-rhamnose at δH 5.10 and the C-2
carbon of L-arabinose at δC 74.6, and the H-1 proton of D-galac-
tose at δH 4.44 and the C-6 carbon of D-glucose I at δC 68.8
showed the linkage points of the sugar molecules to each other.
Accordingly, the structure of 1 was formulated as 3-O-β-D-gluco-
pyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyr-
anosyl hederagenin 28-O-β-D-galactopyranosyl-(1 → 6)-β-D-glu-
copyranosyl ester, namely elmalienoside A.
Compound 3 was shown to have the molecular formula C58H94O26
on the basis of the HR ESIMS data at m/z [M + Na]+ = 1229.5948.
Comparison of the 1H and 13C NMR data (see l Tables 1 and 2) of
"
3 with those of 1 showed considerable structural similarity ex-
cept for the presence of one xylose moiety instead of glucose.
The sugar part of 3 was found to consist of five monosaccharide
residues, identified as L-arabinose (δC 103.8/δH 4.32, d, J =
6.0 Hz), L-rhamnose (δC 100.2/δH 5.13, brs), D-xylose (δC 106.3/δH
4.29, d, J = 7.2 Hz), D-glucose (δC 94.8/δH 5.20, d, J = 6.8 Hz), and D-
galactose (δC 101.7/δH 4.44, d, J = 7.2 Hz) from its NMR spectro-
scopic data. In the HMBC spectrum, long-range correlations be-
tween δH 4.32 (H-1 of Ara) and δC 80.1 (C-3 of aglycone), between
δ
H 5.13 (H-1 of Rha) and δC 74.1 (C-2 of Ara), between δH 4.29 (H-
1 of Xyl) and δC 81.6 (C-3 of Rha), between δH 5.20 (H-1 of Glc)
and δC 176.0 (C-28 of aglycone), and between δH 4.44 (H-1 of
Gal) and δC 68.7 (C-6 of Glc) showed the linkage points of the
monosaccharides to each other and to the aglycone. The identity
of each monosaccaride was determined by acid hydrolysis using
TLC and GC‑MS techniques comparing with authentic sugar sam-
ples, in addition to the COSY, NOESY spectral data and literature
reports [27,28]. The alkaline hydrolysis of compound 3 gave the
known compound 9 (sapindoside B). Consequently, compound 3
(elmalienoside C) was determined to be 3-O-β-D-xylopyranosyl-
(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl hed-
eragenin 28-O-β-D-galactopyranosyl-(1 → 6)-β-D-glucopyranosyl
ester.
"
Elmalienoside B (2) (l Fig. 1) exhibited in the HR ESIMS the [M +
Na]+ peak at m/z 1097.5487 consistent with the molecular formu-
"
la C53H86O22. The NMR data (see l Tables 1 and 2) of the aglycone
Antibacterial activity tests of all extracts and pure compounds
"
of 2 were similar to those of compound 1. Acid hydrolysis of 2
with conc. HCl gave hederagenin, L-arabinose, L-rhamnose, D-glu-
cose, and D-galactose moieties whereas alkaline hydrolysis of 2
with 5% KOH in water yielded a known compound 8. The 1H
NMR spectrum of 2 displayed signals for four anomeric protons
at δH 5.20 (d, J = 8.0 Hz, Glc), 5.04 (brs, Rha), 4.44 (d, J = 7.6 Hz,
Gal), and 4.31 (d, J = 5.6 Hz, Ara), which gave correlations in the
HMQC spectrum, with four anomeric carbon signals at δC 94.8,
100.6, 101.8, and 103.6, respectively. The correlations which
were observed in the HMBC spectrum between the anomeric
proton signals and aglycone carbons at δH 4.31 (Ara) and δC 80.0
were evaluated using the MIC method (l Table 3). All identified
compounds (1–12) were obtained from the most active fraction
of C. elmaliensis. According to the results, all pure compounds, in-
cluding the new ones, were found to be very active against both
gram-positive and gram-negative bacteria. Moreover, all com-
pounds of C. elmaliensis showed very strong antibacterial activity
against E. faecalis, their MIC values being even much lower than
those of the standard antibiotic gentamycin. These results are
similar to those of other reports in the literature concerning the
genus Cephalaria [9,10].
Sarıkahya NB and Kırmızıgül S. Antimicrobially Active Hederagenin… Planta Med 2012; 78: 828–833

Original Papers 833
12 Godjevac D, Vajs V, Menkovic N, Tesevic V, Janackovic P, Milosavljevic S.
Flavonoids from flowers of Cephalaria pastricensis and their antiradical
activity. J Serb Chem Soc 2004; 69: 883–886
13 Pasi S, Aligiannis N, Skaltsounis AL, Chinou IB. A new lignan glycoside
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Acknowledgements
!
The authors gratefully acknowledge TUBITAK (107T028), EBIL-
TEM (2008BIL003), and the Research Grant Office of the Ege Uni-
versity (2009FEN080) for financial support and thank Prof. Dr. H.
Sümbül and Assist. Prof. Dr. R.S. Göktürk for the collection and
identification of the plant. TUBITAK, EBILTEM, and the Izmir Hy-
giene Institute are gratefully acknowledged for providing HR
ESIMS, NMR (400 MHz) spectra, and GC‑MS analyses, respective-
ly. Thanks are due to Assoc. Prof. Ü.K. Yavaşoğlu for assistance
with the antimicrobial activity tests. The authors also thank As-
soc. Prof. Dr. Stephen Astley for proofreading the manuscript.
18 Mao Q, Cao D, Jia XS. Studies on the chemical constituents of Lonicera
macranthoides. Acta Pharm Sin 1993; 28: 273–281
19 Mukhamedziev MM, Alimbaeva PK, Gorovits TT, Abubakirov NK. Struc-
ture of the triterpenoid glycoside from Dipsacus azureus, Dipsacoside
B. Khim Prir Soedin 1971; 7: 153–158
Conflict of Interest
!
There is no conflict of interest between the authors.
20 Kawai H, Kuroyanagi M, Umehara K, Ueno A, Satake M. Studies on the
saponins of Lonicera japonica Thunb. Chem Pharm Bull 1988; 36:
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Tommasi N. Cytotoxic saponins from Schefflera rotundifolia. Planta
Med 2004; 70: 960–966
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