Specialized metabolites from the aerial parts of Centaurea polyclada DC.

Article abstract of DOI:10.1016/j.phytochem.2017.07.002

The genus Centaurea L. (Asteraceae) is represented by 200 taxa in the flora of Turkey and several Centaurea species are used as herbal remedies against different conditions. Previous phytochemical investigations on this genus generally revealed the isolation of sesquiterpene lactones and flavonoid derivatives. In our continuous search on Centaurea genus, a phytochemical study was performed on Centaurea polyclada DC., an endemic of West Anatolia. Previously undescribed two sesquiterpene-amino acid conjugates, an elemane and an eudesmane derivative were isolated from the aerial parts of Centaurea polyclada, together with eight known compounds; two elemane derivatives, three flavonoids, a lignan, a phenolic glucoside and a phenylpropanoid glucoside. Structural elucidation of the compounds was based on spectroscopic evidence, including 1D and 2D NMR and high-resolution mass spectrometry, chemical degradation results and reference data comparison. Sesquiterpene-amino acid conjugates are representatives of an unusual group of sesquiterpenes, and elemane-amino acid conjugates are herein reported for the first time in nature.

Full text of DOI:10.1016/j.phytochem.2017.07.002

Phytochemistry 143 (2017) 12e18
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Specialized metabolites from the aerial parts of Centaurea polyclada
DC.
,
*
Serdar Demir a, Canan Karaalp a, Erdal Bedir b
a Department of Pharmaceutical Botany, Faculty of Pharmacy, Ege University, 35100, Bornova, Izmir, Turkey
b Department of Bioengineering, Faculty of Engineering, Izmir Institute of Technology, 35430, Urla, Izmir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
The genus Centaurea L. (Asteraceae) is represented by 200 taxa in the flora of Turkey and several
Centaurea species are used as herbal remedies against different conditions. Previous phytochemical in-
vestigations on this genus generally revealed the isolation of sesquiterpene lactones and flavonoid de-
rivatives. In our continuous search on Centaurea genus, a phytochemical study was performed on
Centaurea polyclada DC., an endemic of West Anatolia.
Received 25 April 2017
Received in revised form
20 June 2017
Accepted 12 July 2017
Previously undescribed two sesquiterpene-amino acid conjugates, an elemane and an eudesmane
derivative were isolated from the aerial parts of Centaurea polyclada, together with eight known com-
pounds; two elemane derivatives, three flavonoids, a lignan, a phenolic glucoside and a phenylpropanoid
glucoside. Structural elucidation of the compounds was based on spectroscopic evidence, including 1D
and 2D NMR and high-resolution mass spectrometry, chemical degradation results and reference data
comparison. Sesquiterpene-amino acid conjugates are representatives of an unusual group of sesqui-
terpenes, and elemane-amino acid conjugates are herein reported for the first time in nature.
© 2017 Elsevier Ltd. All rights reserved.
Keywords:
Centaurea polyclada
Asteraceae
Specialized metabolite
Sesquiterpene
Amino acid conjugate
L-proline
1. Introduction
Fractionation of CHCl3 and n-BuOH extracts from the aerial parts
of C. polyclada led to the isolation of four previously undescribed
Centaurea polyclada DC. (Asteraceae) is an endemic, profusely
branched, perennial plant and naturally distributed in West Ana-
tolia (Uysal, 2012; Wagenitz, 1975). The genus Centaurea L. is rep-
resented by 200 taxa in the flora of Turkey and several Centaurea
species are used as herbal remedies against inflammatory condi-
tions, such as abscesses, asthma, hemorrhoids, peptic ulcer, ma-
laria, common colds, stomach upset and abdominal pain (Akkol
et al., 2009; Ozcelik et al., 2009). Previous studies have confirmed
that this genus is a source of novel bioactive compounds. Phyto-
chemical investigations on this genus generally revealed the
isolation of sesquiterpene lactones, flavonoids, phenyl propanoids,
lignans and phenolic compounds (Astari et al., 2013; Baykan-Erel
et al., 2010; Bruno et al., 2013; Cardona et al., 1997, 1991; Erel
et al., 2011; Gulcemal et al., 2010; Karamenderes et al., 2007a,
2007b; Skaltsa et al., 1999). Composition of the essential oil of
Centaurea polyclada was previously reported (Erel et al., 2013). In
our continuous search on Centaurea species, a phytochemical study
was performed on Centaurea polyclada DC.
specialized metabolites (1e4) together with eight known com-
pounds (5e12). Herein, the isolation and structural elucidation of
compounds 1e4 are described. 1H and 13C-NMR data of compounds
5 and 6 are also provided (Tables 1 and 2).
2. Results and discussion
CHCl3 and n-BuOH extracts from the aerial parts of C. polyclada
was subjected to a series of column chromatographic separation
steps. Purification studies led to the isolation of previously unde-
scribed two elemane sesquiterpene-amino acid conjugates (1 and
2) together with an elemane derivative (3) and an eudesmane type
sesquiterpene (4). The known compounds; two elemane de-
rivatives; 6a,8a,15-trihydroxyelema-1,3,11(13)-trien-12-oic acid (5)
(Cardona et al., 1992) and its methyl ester (6) (Cardona et al., 1992),
three flavonoids; salvigenin (7) (Salan et al., 2001), eupatorin (8)
and 30-methoxy eupatorin (9) (Salan et al., 2001), a lignan; arctiin
(10) (Shoeb et al., 2004), a phenolic glucoside; tachioside (11) (Liu
et al., 2013) and a phenylpropanoid glucoside; syringin (12)
(Konuklugil and Bahadir, 2004) were identified by comparison of
their spectroscopic data with literature values (Fig. 1). Herein, the
* Corresponding author.
E-mail addresses: erdalbedir@iyte.edu.tr, erdalbedir@gmail.com (E. Bedir).
http://dx.doi.org/10.1016/j.phytochem.2017.07.002
0031-9422/© 2017 Elsevier Ltd. All rights reserved.

S. Demir et al. / Phytochemistry 143 (2017) 12e18
13
Table 1
1H NMR data of compounds 1e6 [400 MHz, CD3OD, dH (J in Hz)].a
Position
1
2
3
4
5
6
1
2
5.84
5.01
4.99
5.39
4.98
dd (17.2/10.8)
d (10.8)
d (17.2)
brs
5.73
4.87
4,89
5.34
4.96
dd (17.2/11.2)
d (11.2)b
d (17.2)
brs
5.77
4.94
4,92
5.35
4.97
dd (18/10.8)
d (10.8)
d (18.0)
brs
3.28
1.77
1.67
1.51
1.55
2.19
1.52
3.87
2.23
4.07
2.27
1.09
dd (11.2/4.4)
m
5.76
4.92
4.91
5.33
4.94
dd (18/10.4)
d (10.4)
d (18)
brs
5.76
4.93
4.92
5.34
4.93
dd (18/10.4)
d (10.4)
d (18)
brs
ddd (12.8/4.4)
3
mb
brs
brs
brs
mb
brs
brs
4
5
6
7
8
9
mb
2.34
4.56
2.03
4.08
1.80
1.61
d (12)
1.89
3.90
1.80
3.93
1.69
1.50
d (10.8)
mb
1.92
4.26
2.64
5.53
1.86
1.62
d (10.8)
t (10.4)
t (10.8)
td (11.2/4.4)
dd (12.4/4.4)
dd (12/11.6)
mb
1.87
4.10
2.33
4.08
1.73
1.50
d (10.8)
1.85
4.13
2.32
4.11
1.74
1.50
d (10.8)
dd (10.8/10.4)
t (10.4)
td (10.8/4.4)
dd (12.8/4.4)
t (12)
dd (11.6/10.8)
ddd (11.6/10.8)
td (10.8/4.4)
dd (13.2/4.4)
dd (13.2/10.8)
t (10.4)
t (10.4)
td (10.8/4.8)
dd (12.4/4.8)
dd (12.4/11.6)
dd (10.4/10)
dd (10.4/10)
td (10.4/4)
dd (12.4/4)
t (12.0)
m
mb
dd (12.4/4)
dd (12.4/12)
10
11
12
13
3.30
mb
3.25
mb
3.66
3.44
1.13
4.03
3.93
dd (13.2/5.2)
dd (13.2/8)
s
d (15.2)
d (15.2)
3.88
3.10
1.05
3.98
3.88
mb
6.28
5.70
1.21
4.01
3.91
brs
brs
s
d (14.8)
d (14.8)
6.33
5.70
0.95
9.31
d (1.2)
d (1.2)
s
6.22
5.62
1.11
3.99
3.89
brs
brs
s
d (14.8)
d (14.8)
6.34
5.75
1.13
3.99
3.89
d (1.6)
d (1.6)
s
d (14.4)
d (14.4)
mb
14
15
s
d (14.4)
d (5.2)
d (14.4)b
10
20
30
3.99
2.44
2.26
2.12
1.93
3.78
3.24
dd (9.2/4)
3.87
2.40
2.20
2.10
1.97
3.75
3.10
mb
m
m
m
m
m
m
m
m
m
mb
6.30
1.97
qd (7.2/1.2)
dd (7.2/1.2)
40
50
m
4.16
4.10
dd (13.6/1.2)
dd (13.6/1.2)
mb
-OCH3
3.74
s
3.76
s
a
Assignments of 1H and 13C NMR data are based on 1H-1H COSY, HSQC and HMBC experiments.
Overlapping signals.
b
Table 2
1H, 13C-NMR and HSQC spectra of 1 revealed a mono-substituted
olefinic system (dH 5.84, 5.01 and 4.99; dC 148.4 and 112.5) sug-
gesting an isolated ethenyl group, a pair of broad singlet protons (dH
5.39 and 4.98, each 1H; dC 112.9) implying an exocyclic double
bond, two geminal oxymethylene protons as AB system doublets
13C NMR data of compounds 1e6 [100 MHz, CD3OD, dC (ppm)].a
Position Type
1
2
3
4
5
6
1
2
3
4
5
6
7
8
CH
148.4
149.1
111.3
112.4
148.5
57.1
70.5
55.1
67.4
49.8
148.5
111.8
112.0
148.1
56.6
72.0
56.9
72.0
44.8
78.3
28.8
25.4
149.2 149.0
111.3 111.4
11.9 112.0
CH2 112.5
CH2 112.9
C
CH
CH
CH
CH
(dH 4.03 and 3.93; dC 66.8), two oxymethine groups (dH 4.56 and
146.2
51.6
80.7
58.0
67.6
49.9 (CH) 148.5 148.3
4.08; dC 80.7 and 67.6) and a tertiary methyl signal (dH 1.13, 3H; dC
19.1) (Fig. 1, Tables 1 and 2). The covalent connectivity of 1 was
established by thorough analysis of the COSYand HSQC spectra. The
association of partial structures and assignment of quaternary
carbon atoms were completed by means of long-range correlations
in the HMBC spectrum.
Based on the COSY and HMBC correlations (Fig. 2), an isolated
framework was readily deduced that was consistent with proline
moiety (dH 3.99, dd, J ¼ 9.2, 4.0 Hz, H-20; 2.44 and 2.26, m, H2-30;
2.12 and 1.93, m, H2-40; 3.78 and 3.24, m, H2-50; dC 174.0, C-10; 72.0,
C-20; 30.5, C-30; 24.8, C-40 and 57.1, C-50) (Matsuda et al., 2000).
After subtracting 5 carbon resonances of the proline residue and
two degrees of unsaturation, the remaining 15 signals were
attributable to a bicyclic sesquiterpene skeleton with three multi-
ple bonds.
53.0
71.4
61.6
68.1
46.5
39.8
139.8
168.5
130.0
12.6
56.7 56.7
72.5 71.6
59.7 60.6
68.9 68.2
49.8 49.6
41.0 41.0
142.5 140.2
166.8 168.7
125.4 129.4
19.4 19.4
9
CH2 49.8
10
11
12
13
14
15
10
C
C
C
43.0
41.0
41.2
44.5 (CH) 41.3 (CH) 140.8
176.8
180.3
57.3
19.4
67.7
173.7
72.1
30.1
24.6
53.6
170.4
128.7
19.1
67.6
CH2 56.6
CH3 19.1
CH2 66.8
C
CH
CH2 30.5
CH2 24.8
CH2 57.1
204.9 (CH) 67.7 67.6
174.0
72.0
167.5
20
133.6 (C)
139.1 (CH)
15.7 (CH3)
63.9
30
40
50
-OCH3
52.0
52.1
A detailed examination of the 1H NMR spectrum of 1 (dH 5.84,
dd, J ¼ 17.2, 10.8 Hz, H-1; 5.01, d, J ¼ 10.8 Hz and 4.99, d, J ¼ 17.2 Hz,
H2-2; 5.39 and 4.98, each br s, H2-3; 4.03 and 3.93, d, J ¼ 15.2 Hz,
H2-15 and 1.13, s, H3-14) displayed characteristic low-field and
high-field signals of an elemane-type framework, which is a com-
mon sesquiterpene skeleton encountered in Centaurea genus
(Cardona et al., 1997; Karamenderes et al., 2007b). The splitting
patterns of the AB system doublets deriving from the hydrox-
ymethyl residue (dH 4.03 and 3.93, JAB ¼ 15.2, H2-15) and the methyl
signal (dH 1.13, s, H3-14) suggested their attachments to quaternary
carbons, whereas the 2JC-H correlations between H2-15 and C-4 (dC
146.2), and H3-14 and C-10 (dC 43.0) in the HMBC spectrum put
a
Assignments of 1H and 13C NMR data are based on 1H-1H COSY, HSQC and HMBC
experiments.
isolation and structural elucidation of compounds 1e4 are
described. NMR data for compounds 5 and 6 were also provided.
Compound 1 was obtained as a yellowish gum. QTOF-MS anal-
ysis displayed protonated and deprotonated molecular ion peaks at
380.2028 [MþH]þ and 378.1921 [M-H]- for positive and negative
modes, suggesting a molecular formula of C20H29NO6. Of the seven
degrees of unsaturation deduced from the molecular formula, four
were present as multiple bonds including two carbonyl carbons (dC
176.8, 174.0, 148.4, 146.2, 112.9 and 112.5). Initial inspection of the

14
S. Demir et al. / Phytochemistry 143 (2017) 12e18
forward the positions of this two groups. Also the allylic coupling
quantity compared to 1. Treatment of 2 with 1% aqueous hydro-
chloric acid (HCl) at room temperature liberated 6 and proline.
Optical rotation measurement of purified proline made clear that
between exomethylene protons at dH 5.39 and 4.98 (H2-3) with H2-
3
15 in the COSY spectrum, and the long-range JC-H correlations
between C-3/H-5 and H-15; C-15/H-3 and H-5; C-5/H-3, H-7 and
H3-14 confirmed their positions in accordance with the elemane
structure.
its absolute configuration was L (½a D20
ꢀ
-85, c. 0.001, MeOH)
(Convention, 2005). Compound 10s proline residue was assumed as
L by way of analogy with 2, whereas the configuration of H-11 in 1
The
a-methylene-g-lactone system is a common structural
was also proposed to be b by means of same inference.
feature for the sesquiterpenes isolated from the genus Centaurea
(Bruno et al., 2013). In the case of compound 1, comparison of the
1H and 13C NMR spectra with previously reported spectroscopic
data of related metabolites revealed that the low-field shift of the
oxymethine proton assigned to H-6 (dH 4.56) indicated an acylation
Molecular formula of 3 was determined as C20H28O7 due to the
sodium adduct and deprotonated ion peaks at m/z 403.1726
[MþNa]þ and 379.1590 [M-H]- obtained by QTOF-MS analyses.
Close examination of the 1H, 13C-NMR and HSQC spectra of 3 once
again suggested an elemane-type structure (dH 5.77, dd, J ¼ 18,
10.8 Hz, H-1; 4.94, d, J ¼ 10.8 Hz and 4.92, d, J ¼ 18 Hz, H2-2; 5.35
and 4.97, each br s, H2-3; 4.01 and 3.91, d, J ¼ 14.8 Hz, H2-15 and
1.21, s, H3-14). When compared to 1 and 2, characteristic exocyclic
at this position, compatible with the 6,12-g-lactone moiety. On the
other hand, the high-field shift of the carbon resonance assigned to
C-11 (dC 44.5) and the COSY correlations of H-7/H-11/H2-13
implied the saturation of the characteristic exocyclic double bond
extending from lactone ring (C-11) (Matsuda et al., 2000). This
prediction was subsequently confirmed by the heteronuclear long-
range correlations of the methine (dC 58.0 and 44.5, C-7 and C-11,
respectively) and carbonyl (dC 176.8, C-12) resonances with the
methylene protons at dH 3.66 and 3.44 assigned to H2-13. Chemical
shift of H2-13 also suggested its connection to an electron with-
drawing group. In the HMBC spectrum, the long-range correlations
between C-20/C-50 (dC 72.0 and 57.1, respectively) and H2-13 protons
not only verified the presence of abovementioned electronegative
group but also linkage site of the proline moiety.
methylene (a-methylene) protons were present as a pair of broad
singlet signals at dH 6.28 and 5.70 (H2-13) in the 1H NMR spectrum.
On the other hand loss of a methine group and presence of an
olefinic carbon resonance referred to C-11 (dC 140.8) were notable.
The location of the exocyclic double bond was straightforwardly
located by the long-range correlations between H2-13 protons and
C-7, C-11 and C-12 (dC 56.9, 140.8 and 170.4, respectively).
Subtraction of the 15 carbon resonances and proton signals
referring to the elemane framework revealed additional resonances
including an olefinic proton (dH 6.30; dC 139.1; C-30), a secondary
methyl signal (dH 1.97; dC 15.7; C-40), an oxymethylene group (dH
4.16 and 4.10; dC 63.9; C-50) and two carbon resonances related to
these groups (a carbonyl and an olefinic at dC 167.5 and 133.6; C-10
and C-20, respectively). The correlations between H-30 and H-40
The relative stereochemistry of 1 was proposed by analyzing the
coupling constants and comparison of the spectroscopic data with
previously reported similar metabolites. Evaluation of the other
naturally occurring elemane-type sesquiterpenes reported from
0
0
(J3 ,4 ¼ 7.2 Hz) in the COSY spectrum and the allylic couplings
0
0
0
0
the genus Centaurea, H-7 was readily assigned with
(Bruno et al., 2013; Karamenderes et al., 2007b). Based on this
postulation, H-6 must be -oriented as the coupling constant sug-
a-configuration
observed for J3 ,5 and J4 ,5 , (each 1.2 Hz) were evident for the
presence of (E)-2-hydroxymethyl-but-2-enoyl (syn. sarracenyl) (Gil
et al., 1992) moiety, a common side chain encountered in Centaurea
sesquiterpenes (Youssef, 1998).
b
gested an antiperiplanar orientation (J6,7 ¼ 11.6 Hz). Moreover, the
coupling constant between H-6 and H-5 protons was also large
(J5,6 ¼ 12 Hz), indicating that H-5 and H-6 were trans-diaxially
When compared to 1 and 2, the low-field shift (ca 1.5 ppm) of
the proton at dH 5.53 (J ¼ 11.2 and 4.4 Hz) assigned to the H-8 was
not only indicated an acylation at this position but also attachment
of the side chain at C-8. The location of the 2-hydroxymethyl-but-
oriented. These results suggested that 1 had H-5(a)/H-6(b)/H-
7(
structure of 1 was established as 13-N-proline-8
elema-1,3-diene-6 ,12-olide, and named as 13-N-proline meli-
a)/H-8(
b
) configurations. On the basis of these findings, the
3
a
,15-dihydroxy-
2-enoyl moiety was directly confirmed by the JC-H correlation
a
between C-10 (dC 167.5) and H-8 (dH 5.53) in the HMBC spectrum.
The relative configurations of the stereocenters in 3 were
identical with those of 1. Analysis of the coupling constants be-
tween H-5/H-6 (J5,6 ¼ 10.8 Hz), H-6/H-7 (J6,7 ¼ 10.4 Hz) and H-7/H-
tensin in accordance with previously reported secondary metabo-
lite melitensin (Bruno et al., 2013).
Compound 2 was isolated as a colorless gum. QTOF-MS spectra
of 2 exhibited protonated and deprotonated molecular ion peaks
for [MþH]þ and [M-H]- at m/z 398.1655 and 396.2018, respectively,
in agreement with the molecular formula C20H31NO7, which was 18
amu greater than compound 1. The 1H and 13C NMR spectra of 2
revealed the similarity of structures when compared to those of 1.
The spectroscopic assignment was completed comprehensively by
examining COSY, HSQC and HMBC spectra. In the case of compound
2 the only difference observed was the high-field shift of a methine
proton (ca. 0.7 ppm) and a carbon (ca. 10 ppm) signal assigned to
C(H)-6 (dH 3.90 and dC 70.5), and these observations suggested a
free hydroxyl group at C-6. Additionally, 6 degrees of unsaturation
deduced from the molecular formula revealed the monocyclic na-
ture of the structure. All these data clearly proved the absence of
8 (J7,8 ¼ 11.2 Hz) suggested that 3 had H-5(
a)/H-6(b)/H-7(a)/H-8(b)
configurations. Based on these findings, the structure of 3 was
established as ,15-
-O-[20-(hydroxymethyl)-but-20-enoyl]-6
dihydroxyelema-1,3,11(13)-trien-12-oic acid.
8
a
a
QTOF-MS analysis of 4 suggested a molecular formula of
C
16H24O6 (m/z 335.1466 [MþNa]þ). 1D and 2D-NMR spectra of 4
revealed one carbonyl
(dC 168.5), two quaternary carbons
(including one olefinic at dC 139.8), one aldehyde (dH 9.31; dC 204.9),
one exocyclic methylene as a pair of broad singlet signals (dH 6.33
and 5.70; dC 130.0), three oxymethine groups (dH 4.07, 3.87 and
3.28; dC 68.1, 71.4 and 78.3), a singlet proton signal attributed to a
methoxy group (dH 3.74, 3H; dC 52.0), three methines (dH 2.23, 2.19
and 2.19), three methylene groups (resonating between dH 1.09 and
2.27; dC 46.5, 28.8 and 25.4) and a methyl (dH 0.95, 3H; dC 12.6).
After subtraction of the methoxy group, the presence of 15
carbon resonances together with overall spectral profile suggested
another sesquiterpene structure. The proton and carbon data
secured by COSY, HSQC and HMBC spectra revealed a different core
structure consistent with an eudesmane framework (Saroglou
et al., 2005) (Fig. 2). Comparison of the spectroscopic data with
those reported in the literature proved the presence of an aldehyde
group at C-15 (dH 9.31, d; dC 204.9) rather than a primary alcohol.
6,12-g-lactone ring in accordance with the aforementioned 18 amu
difference. The relative stereochemistry of compound 2 was based
on 2D-NOESY data. Particularly, the strong NOESY correlation be-
tween H-6 and H-11 allowed us to locate H-11 at
molecule.
b face of the
Consequently, the structure of 2 was established as 13-N-pro-
line-6 ,8 ,15-trihydroxy elema-1,3-diene-12-oic acid.
a
a
To determine the absolute configuration of proline, compound 2
was taken into acidic hydrolysis study because of its higher

S. Demir et al. / Phytochemistry 143 (2017) 12e18
15
Fig. 1. Structures of compounds 1e12.

16
S. Demir et al. / Phytochemistry 143 (2017) 12e18
Fig. 2. COSY and key HMBC correlations of compounds 1 and 4.
The
a
configuration of C-15 was determined based on the up-field
(Marco et al., 2005) was reported in the genus Centaurea.
To the best of our knowledge, the previously undescribed
compounds, 13-N-proline melitensin (1) and 13-N-proline-
shift of the H-15 and its coupling constant with H-4 (J4,15 ¼ 5.2 Hz).
In the case of 15 -CHO, the aldehyde proton was reported to be a
broad singlet resonating at about dH 9.91e9.94 (Bruno et al., 2013).
Presence of a free -oriented hydroxyl group at C-1 was also
ensured by comparing the spectral data of 4 with the literature
(Karioti et al., 2002; Saroglou et al., 2005; Skaltsa et al., 2000).
As in 3, compound 4 provided characteristic signals for an
acrylic acid moiety extending from C-7 and a free hydroxyl group at
C-6 instead of a lactone ring. A weak allylic coupling between
exocyclic methylene protons of C-13 and H-7 in the COSY spectrum,
and the long-range correlations from C-12 to H-7 and H2-13, C-11 to
H-7 and H2-13, and C-13 to H-7 in the HMBC spectrum were
b
6a,8a,15-trihydroxyelema-1,3-dien-12-oic acid (2), are very un-
b
usual representatives of a group of sesquiterpenes containing
amino acid residues, namely L-proline. Elemane-amino acid con-
jugates were encountered for the first time in nature. 8
(hydroxymethyl)-but-20-enoyl]-6
,15-dihydroxyelema-1,3,11(13)-
trien-12-oic acid (3) and methyl ,6 ,8 -trihydroxy-15-oxo-
a
-O-[20-
a
1
b a a
eudesm-11(13)-en-12-oate (4) are also previously undescribed
natural products with elemane and eudesmane-type sesquiterpene
frameworks. On the other hand,
6a,8a,15-trihydroxyelema-
1,3,11(13)-trien-12-oic acid (5) is a commercially available elemane
derivative with no report from natural sources; therefore, it should
be regarded as a new natural product.
3
evident for the acrylic acid residue's position. Additionally, JC-H
correlation between C-12 (dC 168.5) and the methyl signal at dH 3.74
revealed the ester nature of 4 (Skaltsa et al., 2000).
The relative stereochemistry of 4 was resolved on the basis of
coupling constant analysis: [H-5/H-6 (J5,6 ¼ 10.8 Hz), H-6/H-7
(J6,7 ¼ 10.4 Hz) and H-7/H-8 (J7,8 ¼ 10.4 Hz)] and comparison of the
spectroscopic data with relevant structures establishing the con-
3. Experimental
3.1. General experimental procedures
figurations as H-5(
Based on these findings, the structure of 4 was determined as
methyl ,6 ,8 -trihydroxy-15-oxo-eudesm-11(13)-en-12-oate,
the deacyl derivative of 4-epi-carmanin previously reported from
Centaurea achaia (Skaltsa et al., 2000).
a)/H-6(b)/H-7(a)/H-8(b) (Karioti et al., 2002).
IR spectra were obtained on a Perkin Elmer Spectrum 100 FT-IR
spectrometer. Mass spectra analysis was carried out on Thermo-
Scientific TSQ Quantum Access Max LC-MS/MS equipped with an
ESI source. High Resolution Mass spectra were obtained on Agilent
6200 Series TOF and 6500 Series QTOF-MS system equipped with
an ESI source. Optical rotation measurements were done on a
Perkin Elmer Model-351 polarimeter in MeOH at 20 ꢁC. 1D and 2D
(COSY, HMBC, HSQC and NOESY) NMR spectra were recorded on
Varian Oxford AS400 spectrometer with TMS as internal standard
at room temperature. 2D NMR spectra were run using standard
Varian pulse programs. Column chromatography was carried out
1b a a
Secondary metabolites of Centaurea species are mainly sesqui-
terpene lactones with guaiane, germacrane, eudesmane and ele-
mane skeletons (Bruno et al., 2013; Cardona et al., 1997;
Karamenderes et al., 2007b; Karioti et al., 2002; Skaltsa et al.,
2000). As a member of the genus Centaurea, C. polyclada afforded
distinctive sesquiterpene structures. Elemanolides obtained from
Centaurea species are generally melitensin, dehydromelitensin and
their derivatives with different ester chains (Bruno et al., 2013).
In regards to Asteraceae family, a few addition products of
aminoacids to the conjugated double bond of sesquiterpene lac-
tones have been reported and the 13-N-substitued sesquiterpene
derivatives appeared to be the characteristic compounds for Saus-
surea species. Saussureamines (A, B, E) from S. lappa (Matsuda et al.,
2000) and pulchellamine C from S. pulchella (Yang et al., 2008) are
the significant representatives of sesquiterpene-amino acid con-
jugates having guaiane, germacrane and eudesmane skeletons and
L-proline as amino acid residue. Several derivatives were also re-
ported from different Asteraceae members as Inula helenium
(Zaima et al., 2014), Ixeris dentata (Cha et al., 2012) and Scorzonera
divaritica (Yang et al., 2016). Only a germacrane derivative, namely
onopordopicrin-valine dimeric adduct from Centaurea aspera
on silica gel 60 (40e63 m
m-Merck), Sephadex LH-20 (GE Health-
care) and Lichroprep RP-C18 (25e40 m
m, Merck) using analytical
grade purity solvents (Merck and Sigma). TLC analyses were carried
out on silica gel 60 F254 and RP-C18 F254s (Merck) pre-coated
aluminum plates. Compounds were detected by UV
(244e366 nm) and vanillin/H2SO4 reagent followed by heating.
Spraying with ninhydrin reagent followed by heating was used to
detect the amino acid residue.
3.2. Plant material
Centaurea polyclada DC. (Asteraceae) was collected from the
roadside of Kayadibi village district, Izmir, Turkey in June 2010
(500 m, 38ꢁ30021.000 N, 27ꢁ14035.600 E). The plant was confirmed by
Assoc. Prof. Serdar Gokhan Senol from Section of Botany,

S. Demir et al. / Phytochemistry 143 (2017) 12e18
17
Department of Biology, Faculty of Science, Ege University and a
voucher specimen (IZEF-5914) was deposited in the Herbarium of
Ege University, Faculty of Pharmacy, Izmir, Turkey.
Fractions B1/30e39 (585 mg) were chromatographed over si-gel
(45 g) with CHCl3:MeOH:H2O (70:30:3, 61:32:7, 64:50:10, 500 ml
each) and subfractions 165e224 were re-chromatographed over si-
gel (10 g) using CHCl3:MeOH:H2O (70:30:3, isocratic, 200 ml) to
afford 1 (13 mg). Subfractions 225e261 (102 mg) were further
purified with CHCl3:MeOH:H2O (61:32:7, isocratic, 300 ml) and
yielded 2 (23 mg).
Fractions B1/40e57 (436 mg) were subjected to Sephadex LH-20
column (40 g) using MeOH (500 ml). Subfractions 28e40 (153 mg)
were dry-loaded and chromatographed over si-gel (10 g) with
CHCl3:MeOH:H2O (80:20:1, 100 ml; 80:20:2, 400 ml and 70:30:3,
100 ml) and 80 fractions were collected. Subfractions 20e28
(10 mg) and 45e80 (34 mg) were separately purified with
CHCl3:MeOH:H2O (80:20:1, isocratic, 100 ml) to afford 11 (7 mg)
and 6 (19 mg) respectively.
3.3. Extraction and isolation
Air-dried and grounded aerial parts of the plant (2.2 kg) were
extracted with n-hexane, chloroform (CHCl3) and methanol
(MeOH) (3 ꢂ 5 L, each) by sonification for 24 h and then filtered.
The extracts were separately evaporated under reduced pressure to
dryness and yielded 44.3 g n-hexane, 35.9 g CHCl3 and 121.3 g
MeOH extracts. Dried MeOH extract was suspended in water and
partitioned with n-butanol (n-BuOH) (H2O: n-BuOH; 1:7) and
yielded 34.5 g n-BuOH extract.
CHCl3 extract (30 g) was dry loaded and chromatographed over
RP-C18 column (200 g) (H2O: MeOH, 80:20 to 0:100; 10%
decreasing polarity, each 1 L) and 30 main fractions (Frs 1e30) were
collected. Based on TLC profiles, 6 main fractions; C1 (Frs. 3e5), C2
(Frs. 8e9), C3 (Frs. 10e12), C4 (Frs. 14e18), C5 (Frs. 19e23) and C6
(Frs. 24e27) were selected for further purification.
Fraction C1 (827 mg) was subjected to Sephadex LH-20 column
(80 g) using 100% MeOH and afforded 60 subfractions. Frs. C2/
16e27 (335 mg) chromatographed over silica gel column (si-gel)
(15 g) using CHCl3:MeOH:H2O (90:10:1, 200 ml; 85:15:0.5 and
80:20:2, 100 ml each) and subfractions 17e28 (143 mg) were re-
chromatographed over si-gel with EtOAc:MeOH:H2O (100:5:0.5,
isocratic, 300 ml). Subfractions 61e75 (76 mg) were further puri-
fied using CHCl3:MeOH:H2O (90:10:1, isocratic, 100 ml) to afford 4
(31 mg).
Fraction C2 (970 mg) was chromatographed over si-gel (25 g)
using CHCl3:MeOH:H2O (90:10:1, 80:20:1 and 70:30:3, 200 ml
each) and 95 sub-fractions were obtained. Frs. 3e11 (285) were
subjected to si-gel (10 g) with n-hexane:EtOAc (60:40 to 50:50,
with 2% increasing polarity, 100 ml each). Subfractions 94e156
(137 mg) were re-chromatographed over si-gel (CHCl3:MeOH:H2O,
90:10:0.5, isocratic, 100 ml) and 5 (38 mg) was obtained.
Fraction C3 (1979 mg) was subjected to si-gel (35 g) with
CHCl3:MeOH:H2O (90:10:1, 400 ml and 80:20:1, 200 ml) and sub-
fraction 39e66 (772 mg) were purified with si-gel (EtOAc:-
MeOH:H2O, 100:5:1, isocratic, 200 ml) to afford 10 (640 mg).
Fraction C4 (2685 mg) was chromatographed over si-gel column
(40 g) using CHCl3:MeOH:H2O (90:10:1, 400 ml and 80:20:1,
200 ml) and finally treated with MeOH. MeOH fraction (212 mg)
was further purified with CHCl3:MeOH:H2O (90:10:1, 200 ml and
85:15:0.5, 300 ml) over si-gel (15 g) and 3 (59 mg) was obtained.
A small amount of residue was observed in the fraction C5.
Insoluble part (788 mg) was centrifuged and chromatographed
over si-gel column (15 g) using CHCl3 (300 ml) to afford 8 (247 mg).
The soluble portion (1300 mg) was chromatographed over si-gel
(25 g) with CHCl3:MeOH:H2O (90:10:1, 300 ml and 70:30:3,
100 ml) and subfractions 5e28 (730 mg) were subjected to
Sephadex LH-20 column (50 g) using MeOH (500 ml). Subfractions
21e31 (51 mg) were further purified over si-gel (10 g) using CHCl3
(100 ml) to yield 9 (9 mg).
Fraction B2 (1375 mg) was chromatographed over Sephadex LH-
20 column (80 g) with MeOH (500 ml) and afforded 150 sub-
fractions. Fractions 19e39 (830 mg) were subjected to RP-C18
column (10 g) using H2O:MeOH (100:0 to 80:20, 2% decreasing
polarity, 100 ml each). The insoluble portion of subfractions
107e145 was centrifuged and 12 (169 mg) was obtained.
3.4. Acid treatment of 13-N-proline-6a,8a,15-trihydroxyelema-1,3-
dien-12-oic acid (2)
Acid treatment of 2 (15 mg) with 1% aqueous hydrochloric acid
(HCl, 10 ml) at room temperature affording a mixture (Matsuda
et al., 2000). An aliquot of the reaction mixture was subjected to
normal phase silica TLC (n-BuOH:Acetic acid:H2O, 3:1:1) to detect
the amino acid by Ninhydrin reagent followed by heating. After
verifying the presence of proline, the mixture was dried and sub-
jected to silica gel column (10 g) with CHCl3:MeOH:H2O (70:30:3
and 61:32:7100 ml each). Subfractions 39e52 were re-
chromatographed over Sephadex LH-20 (10 g) using MeOH
(100 ml) to afford proline (4 mg). Optical rotation measurement
verified the amino acid residue as L-proline (½a D20
ꢀ
-85, c. 0.001,
MeOH) (Convention, 2005).
3.5. Compound characterization
13-N-proline melitensin (1): Yellowish gum; ½a D
ꢀ
20- 17.5 (c. 0.005,
MeOH); 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD,
100 MHz) data: see Tables 1 and 2; QTOF-MS m/z ¼ 380.2028
[MþH]þ (calcd. for C20H30NO6: 380.2073, positive mode) and
378.1921 [M-H]- (calcd. for C20H28NO6: 378.1917, negative mode).
13-N-proline-6
a
,8
a
,15-trihydroxyelema-1,3-dien-12-oic
acid
(2): Colorless gum; ½a D20
ꢀ
- 11.3 (c. 0.008, MeOH); IR (KBr) 3389,
2929, 1590, 1411, 1339, 1208, 1135, 1049, 1008 cmꢃ1 1H NMR
;
(CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data: see
Tables 1 and 2; QTOF-MS m/z ¼ 398.1655 [MþH]þ (calcd. for
C
20H32NO7: 398.2179, positive mode) and 396.2018 [M-H]- (calcd.
for C20H30NO7: 396.2022, negative mode).
-O-[20-(hydroxymethyl)-but-20-enoyl]-6
dihydroxyelema-1,3,11(13)-trien-12-oic acid (3): Yellowish gum;
a 2D0
þ27 (c. 0.003, MeOH); IR (KBr) 3405, 2931, 1702, 1396, 1238,
1165, 1052, 1002, 915 cmꢃ1 1H NMR (CD3OD, 400 MHz) and 13C
8a
a,15-
Fraction C6 (1.4 g) was subjected to Sephadex LH-20 column
(80 g) with MeOH (600 ml) and 120 subfractions were obtained.
Subfractions 84e120 (78 mg) were chromatographed over si-gel
column (CHCl3, 200 ml) and yielded 7 (35 mg).
n-BuOH extract (30 g) was dry loaded and chromatographed
over RP-C18 column (200 g) using H2O:MeOH (100:0 to 0:100; 10%
decreasing polarity, each 1 L). 30 main fractions (Frs 1e30) were
collected and based on TLC profiles, 2 main fractions; B1 (Frs. 2e6)
and B2 (Frs. 7e8) were selected for further purification.
½ ꢀ
;
NMR (CD3OD, 100 MHz) data: see Tables 1 and 2; QTOF-MS m/
z ¼ 403.1726 [MþNa]þ (calcd. for C20H28O7Na: 403.1733, positive
mode) and 379.1590 [M-H]- (calcd. for C20H27O7: 379.1757, negative
mode).
Methyl 1b,6a,8a-trihydroxy-15-oxo-eudesm-11(13)-en-12-oate
(4): Yellowish gum; ½a D20
ꢀ
- 15 (c. 0.003, MeOH); IR (KBr) 3405,
2949, 2353, 1712, 1626, 1442, 1334, 1262, 1198, 1167, 1033 cmꢃ1; 1H
NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data: see
Tables 1 and 2; QTOF-MS m/z ¼ 335.1466 [MþNa]þ (calcd. for
Fraction B1 (6 g) was chromatographed over RP-C18 (100 g)
using H2O (500 ml) and 100 fractions were collected.

18
S. Demir et al. / Phytochemistry 143 (2017) 12e18
Secondary metabolites of Centaurea calolepis and evaluation of cnicin for anti-
C16H24O6Na: 335.1471, positive mode).
inflammatory, antioxidant, and cytotoxic activities. Pharm. Biol. 49, 840e849.
Gil, R.R., Pacciaroni, A.D.V., Oberti, J.C., Diaz, J.G., Herz, W., 1992. A rearranged ger-
macranolide and other sesquiterpene lactones from Stevia jujuyensis. Phyto-
chemistry 31, 593e596.
6a
,8a
,15-trihydroxyelema-1,3,11(13)-trien-12-oic
acid
(5):
Yellowish gum; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD,
100 MHz) data: see Tables 1 and 2; QTOF-MS m/z ¼ 587.2832
[2MþNa]þ (calcd. for C30H44O10Na: 587.2832, positive mode) and
281.1371 [M-H]- (calcd. for C15H21O5: 281.1389, negative mode).
Gulcemal, D., Alankus-Caliskan, O., Karaalp, C., Ors, A.U., Ballar, P., Bedird, E., 2010.
Phenolic glycosides with antiproteasomal activity from Centaurea urvillei DC.
subsp urvillei. Carbohyd Res. 345, 2529e2533.
Karamenderes, C., Bedir, E., Abou-Gazar, H., Khan, I.A., 2007a. Chemical constituents
of Centaurea cadmea. Chem. Nat. Comp. 43, 694e695.
Karamenderes, C., Bedir, E., Pawar, R., Baykan, S., Khan, K.A., 2007b. Elemanolide
sesquiterpenes and eudesmane sesquiterpene glycosides from Centaurea hier-
apolitana. Phytochemistry 68, 609e615.
Karioti, A., Skaltsa, H., Lazari, D., Sokovic, M., Garcia, B., Harvala, C., 2002. Secondary
metabolites from Centaurea deusta with antimicrobial activity. Z Naturforsch C
57, 75e80.
Methyl 6a,8a,15-trihydroxyelema-1,3,11(13)-trien-12-oate (6):
Yellowish gum; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD,
100 MHz) data: see Tables 1 and 2; QTOF-MS m/z ¼ 319.1518
[MþNa]þ (calcd. for C16H24O5Na: 319.1521, positive mode).
Acknowledgments
Konuklugil, B., Bahadir, O., 2004. Phenylpropanoid glycosides from Linum olympi-
cum (Linaceae). Turk J. Chem. 28, 741e744.
Liu, S.K., Que, S., Cheng, W., Zhang, Q.Y., Liang, H., 2013. Chemical constituents from
whole plants of Carduus acanthoides. Zhongguo Zhong Yao Za Zhi 38,
2334e2337.
Marco, J.A., Sanz-Cervera, J.F., Yuste, A., Sancenon, F., Carda, M., 2005. Sesquiter-
penes from Centaurea aspera. Phytochemistry 66, 1644e1650.
Matsuda, H., Kageura, T., Inoue, Y., Morikawa, T., Yoshikawa, M., 2000. Absolute
stereostructures and syntheses of saussureamines A, B, C, D and E, amino acid-
sesquiterpene conjugates with gastroprotective effect, from the roots of Saus-
surea lappa. Tetrahedron 56, 7763e7777.
The authors would like to thank Assoc. Prof. Serdar Gokhan
Senol for authenticating the plant material, and Dr. Salih Gunnaz for
all NMR measurements. This research project was supported by the
Scientific Research Projects Directorate of Ege University (Project
No: 12-ECZ-009).
Appendix A. Supplementary data
Ozcelik, B., Gurbuz, I., Karaoglu, T., Yesilada, E., 2009. Antiviral and antimicrobial
activities of three sesquiterpene lactones from Centaurea solstitialis L. ssp sol-
stitialis. Microbiol. Res. 164, 545e552.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.phytochem.2017.07.002.
€
Salan, U., Topçu, G., Oksüz, S., 2001. Flavonoids of Centaurea kilaea and C. salonitana.
J. Fac. Pharm. Istanb. 34, 55e61.
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