Amino acid-sesquiterpene lactone conjugates from the aerial parts of Centaurea pungens and evaluation of their antimicrobial activity

Article abstract of DOI:10.1016/j.fitote.2018.12.001

Two new amino acid-sesquiterpene lactone conjugates, named centaureolide A (1) and centaureolide B (2) along with a germacrane derivative (3), five flavonoids (4–8) and one quinic acid derivative (9) have been isolated from the aerial parts of Centaurea pungens (Asteraceae). Their structures were established by a combination of one- and two-dimensional NMR techniques, and mass spectrometry. The never reported sesquiterpene lactones have been determined as germacrane derivatives (1 and 2) characterized by the unusual occurrence of a proline moiety. In order to validate the use of the extract of C. pungens in folk medicine, the antimicrobial activity of the isolated compounds against the Gram-positive strains Bacillus cereus, Staphylococcus aureus and Listeria innocua and the Gram-negative strains Pseudomonas aeruginosa and Pseudomonas fragi was evaluated.

Full text of DOI:10.1016/j.fitote.2018.12.001

Fitoterapia 133 (2019) 51–55
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Amino acid-sesquiterpene lactone conjugates from the aerial parts of
Centaurea pungens and evaluation of their antimicrobial activity
Fatiha Labeda,1, Milena Masullo , Virginia Mirra , Filomena Nazzaro , Fadila Benayache ,
b,1
b
c
a
a
b,⁎
Samir Benayache , Sonia Piacente
a
Unité de recherche Valorisation des Ressources Naturelles, Molécules Bioactives et Analyses Physicochimiques et Biologiques, Université des Frères Mentouri Constantine,
Algeria
Università degli Studi di Salerno, Dipartimento di Farmacia, Via Giovanni Paolo II, 132, 84084 Fisciano (SA), Italy
b
c
Istituto di Scienze dell'Alimentazione CNR-ISA, Via Roma 64, 83100 Avellino, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two new amino acid-sesquiterpene lactone conjugates, named centaureolide A (1) and centaureolide B (2) along
with a germacrane derivative (3), five flavonoids (4–8) and one quinic acid derivative (9) have been isolated
from the aerial parts of Centaurea pungens (Asteraceae). Their structures were established by a combination of
one- and two-dimensional NMR techniques, and mass spectrometry. The never reported sesquiterpene lactones
have been determined as germacrane derivatives (1 and 2) characterized by the unusual occurrence of a proline
moiety. In order to validate the use of the extract of C. pungens in folk medicine, the antimicrobial activity of the
isolated compounds against the Gram-positive strains Bacillus cereus, Staphylococcus aureus and Listeria innocua
and the Gram-negative strains Pseudomonas aeruginosa and Pseudomonas fragi was evaluated.
Amino acid-sesquiterpene lactones
Germacrane derivatives
Centaurea genus
Asteraceae
Antimicrobial activity
1. Introduction
structural elucidation of two germacrane derivatives (1–2), never re-
ported in literature, along with seven known compounds among which
The genus Centaurea (tribe Cynareae, family Asteraceae), re-
one germacranolide (3), five flavonoids (4–8) and one quinic acid de-
rivative (9) has been reported. Their structures were established by a
combination of one- and two-dimensional NMR techniques, along with
mass spectrometry. The two previously unreported germacrane deri-
vatives (1 and 2) are characterized by the unusual occurrence of a
proline moiety. In order to validate the use of the extract of C. pungens,
and on the basis of the antimicrobial activity reported for germacrane
derivatives [8], the antimicrobial activity of the isolated compounds
against the Gram-positive strains Bacillus cereus, Staphylococcus aureus,
and Listeria innocua and the Gram-negative strains Pseudomonas aeru-
ginosa, and Pseudomonas fragi was evaluated.
presented by herbaceous thistle-like flowering plants, is widely dis-
tributed all over the world [1]. It includes more than 500 species, 45 of
which growing spontaneously in Algeria, with 7 species localized in the
Sahara desert [2]. Many Centaurea species are used in the folk medicine
of many countries for treatment of cancer, microbial infections and as
stimulant, tonic, diuretic and anti-rheumatic remedy [3,4]. The main
secondary metabolites of Centaurea species are represented by tri-
terpenes, flavonoids, lignans, and sesquiterpene lactones [3,5,6].
C. pungens is a herbaceous plant with spined bracts on the flower-
head, of rocky and sandy pasturages in Algeria and Mauritania, oc-
curring throughout the Sahara area. It provides good grazing for all
stock. Moreover, this species is used as condiment and in folk medicine
for the treatment of respiratory infections [7].
2. Results and discussion
To the best of our knowledge, there is no report on the secondary
metabolites of C. pungens and their biological activity. In the frame of
our ongoing researches on Algerian plants, the phytochemical study of
the aerial parts of C. pungens was carried out with the aim to investigate
the chemical composition and to validate the antimicrobial activity
attributed to this plant by folk medicine. Herein, the isolation and
2
The MeOH/H O extract of the flowers of C. pungens was fractionated
by chromatographic methods, to afford two germacrane derivatives
never reported in literature (1–2), one germacranolide (3), five flavo-
noids (4–8) and one quinic acid derivative (9) (Fig. 1). Their structures
were established by 1D and 2D NMR experiments in combination with
HR-ESIMS analyses (Table 1).
⁎
Corresponding author.
E-mail address: piacente@unisa.it (S. Piacente).
These authors contributed equally to the work
1
https://doi.org/10.1016/j.fitote.2018.12.001
Received 4 August 2018; Received in revised form 30 November 2018; Accepted 2 December 2018
Available online 14 December 2018
0367-326X/ © 2018 Elsevier B.V. All rights reserved.

F. Labed et al.
Fitoterapia 133 (2019) 51–55
1
OH
H
OH
O
1
0
8
2
O
5
H
1
3
COOH
6
4
7
1
COOH
11
N
H
O
HO
O
N
HO
HO
O
O
O
HOH
2
C
O
1
2
HO
HO
3
OH
OH
OH
1
R OOC
O
HO
HO
O
R
1
O
O
O
O
O
OH
R
2
3
H CO
OH
HOOC
OH
R1
R1
R
2
O
4
5
CH3
H
OH
H
7
8
H
CH3
O
OH
HO
6
H
OH
GlcA
9
HO
Fig. 1. Chemical structures of secondary metabolites isolated from Centaurea pungens.
Table 1
(d, J = 9.6), assigned to H-1 and H-5 of the germacrane moiety, at δ
4.23 and 4.00 (each, d, J = 13.3 Hz), ascribable to a primary alcoholic
function, at δ 5.16 (t, J = 9.6 Hz) and 4.05 (brdd, J = 3.2, 12.0 Hz),
typical of protons linked to oxymethine carbons, and at δ 1.44 (s)
corresponding to a vinyl methyl group. The primary alcoholic function
was assigned to C-15 on the basis of the HMBC correlations between the
proton signals at δ 4.23 and 4.00 with the carbon resonances at δ 144.3
1
H (600 MHz) and 13C (150 MHz) NMR spectroscopic data for compounds 1–2.
1
2
δ
C
δ
H
(J in Hz)
δ
C
H
δ (J in Hz)
1
2
3
4
5
6
7
8
9
129.4
26.6
35.3
144.3
129.2
78.1
58.3
71.8
52.6
5.00, dd (5.9, 10.6)
2.20 (2H), m
2.62, 1.95, m
–
4.82, d (9.6)
5.16, t (9.6)
2.35, td (9.6, 3.2)
4.05, dd (3.2, 12.0)
2.62, 2.40, m
131.2
26.0
34.6
139.2
135.0
68.9
58.4
81.3
47.7
5.05, brd (10.7)
2.31, 2.20, m
2.63, 1.99, m
–
4.97, d (9.3)
4.53, t (9.3)
2.55, m
(
C-4), 129.2 (C-5) and 35.3 (C-3). The alcoholic function at δ 4.05 was
located at C-8 by COSY and HMBC correlations. The presence of an
oxygenated function at C-6α (δ 78.1) was suggested by the correlation
in the COSY spectrum of proton at δ 5.16 (t, J = 9.6 Hz) with the signals
at δ 4.82 (H-5) and δ 2.35 (H-7). The HMBC correlation between the
proton signal at δ 5.16 (H-6) and the carbon resonance at δ 177.5 (C-12)
allowed us to identify a γ-lactone unit. Further signals at δ 3.67 (dd,
J = 6.6 and 12.9 Hz) and 3.63 (dd, J = 5.5 and 12.9 Hz), along with the
4.34, t (9.6)
2.70, brd (12.4),
2
.56, m
10
11
12
13
134.8
43.9
177.5
57.9
–
139.5
44.4
175.6
58.2
–
3.32, m
–
3.63, dd (5.5, 12.9)
3
1.44, s
4.23, d (13.3)
4
3.37, ddd (2.9, 9.6, 10.0)
–
3.52, dd (10.0, 13.0)
3.62, dd (2.9, 13.0)
1.53, s
4.23, d (12.9)
3.88, d (12.9)
Proline
1
typical spin system of a proline unit were observed in the H NMR
spectrum (Table 1). A detailed analysis of NMR data suggested that
compound 1 possessed a germacrane unit, characterized by the pre-
sence of protons linked to an amminomethylene carbon, instead of the
.67, dd (6.6, 12.9)
1
1
4
5
16.9
60.5
16.5
59.6
13
secondary methyl group occurring at C-13. The C resonance of C-13 at
.00, d (13.3)
δ 57.9 along with key HMBC correlations between the signals at δ 3.67
and 3.63 with the carbon signals at δ 43.9 (C-11), 58.3 (C-7), 177.5 (C-
12), 71.4 (C-2′) and 55.9 (C-5′) allowed to establish the site of linkage
of the proline unit.
Proline
173.2
71.4
29.8
24.5
1′
2′
3′
4′
5′
–
173.2
71.5
29.5
24.3
54.8
–
3.98, dd (3.7, 9.2)
2.47, 2.32, m
2.15, 2.00, m
3.86, 3.23, m
3.94, dd (5.7, 9.2)
2.49, 2.19, m
2.17, 1.99, m
3.88, 3.17, m
The relative stereochemistry of 1 was attributed by analysis of the
coupling constants and ROESY correlations, in comparison with lit-
erature. The β orientation of H-11 was deduced from the coupling
constant (9.6 Hz) between H-7 and H-11, typical of a trans-diaxial
disposition of H-7 and H-11 [10]. The β-orientation of the hydroxy
function at C-8 was deduced by the coupling constant (3.2 Hz) between
H-7 and H-8, and confirmed by the absence of the ROESY correlation
between H-6 and H-8, observable in case of an α-oriented hydroxy
group at C-8 [10]. Therefore, the structure of compound 1 has been
established as depicted in Fig. 1 and named centaureolide A.
55.9
The molecular formula of compound 1 (C20
H
29NO
termined by C NMR and HR-ESIMS data (m/z 378.1925 [M-H]
calcd for C20 28NO
showed a band at 1775 cm
6
) was de-
1
3
−
,
H
6
at m/z 378.1917). The IR spectrum of compound 1
−1
attributed to a γ-lactone group. The
1
13
features of the H and C NMR spectra suggested a germacrane ring
with two double bonds at Δ
trum showed signals at δ 5.00 (dd, J = 5.9 and 10.6 Hz) and at δ 4.82
1
,10
4
and Δ [9]. In detail, the proton spec-
52

F. Labed et al.
Fitoterapia 133 (2019) 51–55
The molecular formula of compound 2 (C20
H
29NO
termined by C NMR and HR-ESIMS data (m/z 378.1922 [M-H]
calcd for C20 28NO
showed a band at 1775 cm
6
) was de-
Table 2
1
3
−
,
Minimal Inhibitory Concentration (MIC, μg/mL) of Compounds 1–9 of
Centaurea pungens.
H
6
at m/z 378.1917). The IR spectrum of compound 2
−
1
indicative of the presence of a γ-lactone
1
2
3
4
5
6
7
8
9
group. The analysis of NMR data of 2 revealed also for this compound a
germacrane skeleton with a γ-lactone group. In particular, the proton
spectrum showed signals at δ 5.05 (brd, J = 10.7 Hz, H-1), 4.97 (d,
J = 9.3, H-5), 4.23 and 3.88 (each, d, J = 12.9 Hz, H-15), 4.53 (t,
J = 9.3 Hz, H-6), 4.34 (t, J = 9.6 Hz, H-8), and at δ 1.53 (3H, s, H-14).
The location of the γ-lactone unit was established on the basis of the
downfield shift of C-8 at δ 81.3 and the upfield shift of C-6 at δ 68.9
revealing the occurrence of a C-8/C-12 olide ring. A detailed analysis of
4
4
PA
PF
LI
384 BCE 1000 1000 160
313 BCE 1000 1000 300
1000 300
1000 300
1000 1000 160
1000
160
100
1000 300
100 160
1000 1000 1000 1000 400
1000 1000 80 40 80
160
100
100
400
400
100
160
100
100
1000 1000 1000
1000 100 1000
4
384 BCE: Bacillus cereus DSM 4384; 4313 BCE: Bacillus cereus 4313; PA:
Pseudomonas aeruginosa; PF: Pseudomonas fragi; LI Listeria innocua.
2
D-NMR (HSQC, HMBC and COSY) experiments revealed that com-
(
MIC = 160 μg/mL).
Hispidulin (5) exhibited the highest antimicrobial effectiveness: the
pound 2 showed chemical features similar to those reported for 11,13-
dihydroartemisiifolin [11], differing from it by replacement of Me-13
presence of 20 μg of hispidulin on the filter paper disk led to the for-
mation of an inhibition halo of about 14 mm in diameter against Listeria
innocua, and a halo of about 8.7 mm against P. aeruginosa, with MIC
values of 40 μg/mL and 100 μg/mL, respectively. In literature the an-
timicrobial activity of hispidulin obtained from Inula viscosa, against B.
cereus [19] and from Cirsium arvense against P. aeruginosa [20] is re-
ported.
with an amminomethylene function (δ
H
C
3.52 and 3.62, δ 58.2). In a
previous study, the conformations of germacranolides, showing broa-
dened NMR spectra or even multiple NMR signals indicative of their
1
13
conformational mobility, were investigated by means of H and
NMR spectroscopy and quantum mechanical calculations [11]. For
1,13-dihydroartemisiifolin a major conformer (in a ratio 85:15) was
C
1
described; its NMR data are in agreement with those of compound 2. As
The antimicrobial activity of sesquiterpene lactones is known as
well as that exhibited by sesquiterpene lactones isolated from Centaurea
genus, especially against P. aeruginosa [20]. Moreover, the activity of
some of the tested compounds against Listeria, in particular hispidulin,
opens appealing perspectives on the use of this compound as a natural
preservative agent also in other fields, such as food science. The results
obtained suggest the possibility of using this compound as a pre-
servative agent of certain foods that, if not processed or stored in an
appropriate manner, could be contaminated by Listeria.
for 11,13-dihydroartemisiifolin, a coupling constant of 9.6 Hz between
H-7 and H-11 and a ROE correlation between H-7 and the CH –13 was
2
observed for compound 2.
Additionally, the proton spectrum of 2 revealed the presence of a
proline unit. Key HMBC correlations between the signals at δ 3.52 and
3
.62 with the carbon signal at δ 71.5 (C-2′) and 54.8 (C-5′) were ob-
served. Therefore, the structure of compound 2 was elucidated as de-
picted in Fig. 1 and named centaureolide B.
To determine the absolute configuration of proline, compound 2
was taken into acid hydrolysis study because of its higher quantity if
compared to 1. The absolute configuration of the proline unit was de-
termined by acid hydrolysis of compound 2 as L- proline and was as-
signed on the basis of its optical rotation value [12].
3. Experimental section
3.1. General instrumentation
To the best of our knowledge, the previously undescribed com-
Optical rotations were measured on a Autopol IV (Rudolph Research
pounds, centaureolides
A (1) and B (2), are very unusual re-
Analytical) polarimeter. IR measurements were obtained on a Bruker
IFS-48 spectrometer. NMR experiments were carried out on a Bruker
DRX-600 spectrometer (Bruker BioSpinGmBH, Rheinstetten, Germany)
equipped with a Bruker 5 mm TCI CryoProbe at 300 K. All 2D-NMR
presentatives of a group of sesquiterpenes containing amino acid re-
sidues, in this case L-proline. A recent study reports an unusual adenine-
substituted germacrane sesquiterpene lactone, isolated from
Elephantopus tomentosus [13]. So far, the only one germacrane linked to
a proline unit is represented by saussureamine A, isolated from Saus-
surea lappa [14]. Noterworthy, compounds 1 and 2 represent the first
report in Centaurea genus of germacrane-amino acid conjugates, fol-
lowing the recent report of elemane-amino acid conjugates from C.
polyclada [12].
spectra were acquired in MeOD
4
(99.95%, Sigma-Aldrich, Munich,
Germany) and standard pulse sequences and phase cycling were used
for DQF-COSY, HSQC, HMBC, and ROESY spectra. The NMR data were
processed using TopSpin 3.2 software. HRESIMS data were acquired on
an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San
Jose, CA, USA) operating in negative ion mode.
Additionally, 11β,13-dihydrosalonitenolide 15-O-β-glucopyrano-
side (3) [15], 6-methoxy,7-methyl-luteolin (4) [16], hispidulin (5)
[
17], hispidulin 7-O-β-D-glucuronopyranoside (6) [18], apigenin-7-O-
3.2. Plant material
β-D-glucuronopyranoside (7) [18], apigenin-7-O-(6-methoxy)-β-D-glu-
curonopyranoside (8) [18] and 5-O-caffeoylquinic acid (9) [17] have
been isolated.
The aerial parts of Centaurea pungens were collected in the flowering
stage on May 2015 from the region of Bechar in the south west of
Algeria and authenticated by Professor Mohamed Kaabeche from the
University of Setif. A voucher specimen CP145/11 has been deposited
in the Herbarium of the VARENBIOMOL research unit, Frères Mentouri
University, Constantine 1.
With the aim to validate the use of C. pungens in folk medicine, the
antimicrobial activity of the isolated compounds against the Gram-po-
sitive strains Bacillus cereus, Staphylococcus aureus, and Listeria innocua
and the Gram-negative strains Pseudomonas aeruginosa, Pseudomonas
fragi was evaluated. Results are shown in Table S1. The Minimal
Inhibitory Concentrations (MIC) are reported in Table 2.
3.3. Extraction and isolation
Pseudomonas aeruginosa was the strain exhibiting the highest sen-
sitivity to all the compounds isolated from C. pungens which resulted
active already at 10 μg/disk, with inhibition halos greater than 4 mm,
and with MIC values not higher than 160 μg/mL. On the contrary,
Pseudomonas fragi showed a greater resistance to the tested compounds,
only compounds 1 and 6 showed a weak inhibitory activity against this
strain, producing an inhibition halo not higher than 4 mm at 20 μg/
disk. Both two strains of Bacillus cereus were sensitive to compound 8
Air-dried flowers (720 g) of Centaurea pungens were extracted at
room temperature with MeOH/H₂O (7:3) for 72 h, three times. The
combined ethanolic extracts were evaporated at room temperature,
suspended in water (400 mL) under magnetic stirring, and kept in the
refrigerator for one night to precipitate a maximum of chlorophylls.
After filtration, the resulting solution was successively partitioned with
3
CHCl , EtOAc and n-BuOH. The organic phases were dried with
53

F. Labed et al.
Fitoterapia 133 (2019) 51–55
Na
2
SO
4
, filtered and concentrated in vacuum up to 35 °C to obtain the
(3.54 g), EtOAc (5.22 g) and n-BuOH
16.41 g). A part of the EtOAc extract (4.5 g) was fractioned on silica gel
Milano, Italy) were used as negative and positive control, respectively.
The samples were tested in triplicate, and the results are expressed as
the mean values ± standard deviations.
following extracts: CHCl
(
3
column eluted with CHCl
3
/acetone step gradients to yield 30 fractions
/acetone, 94:6) (58 mg) and
/acetone, 92:8) (191 mg) gave precipitates which
were filtered and washed with methanol to afford compound 5
monitored by TLC. Fraction 5 (CHCl
fraction 9 (CHCl
3
3.5.2. Minimum inhibitory concentration
3
The MIC values for samples 1–9 were evaluated by the resazurin
microtiter-plate assay [23]. Samples were dissolved in DMSO and dis-
tributed in a multiwell plate with different volumes of Muller-Hinton
broth (Sigma-Aldrich, Milano, Italy). Twofold serial dilutions were
performed such that each well had 50 μL of the test material in serially
descending concentrations. A 35 μL amount of 3.3× strength iso-
sensitized broth and 5 μL of resazurin, used as indicator solution, were
added to a final volume/well of 240 μL. Finally, 20 μL of bacterial
suspension was added to each well to achieve a concentration of about
(
39.6 mg) and compound
4
(20.8 mg), respectively. Fraction 17
/MeOH
/MeOH,
2:8) showed the formation of a precipitate, which was filtered and
washed with MeOH to afford compound 7 (8.6 mg). From fraction 26
265.5 mg) (CHCl /acetone, 55:45), an aliquot was purified on a
(
1300 mg) was subjected to a silica gel 60 column using CHCl
3
as eluent, to give 20 subtractions (1−20). Subfraction 9 (CHCl
9
3
(
3
Sephadex LH20 column eluted with MeOH, to yield compound 2 (5 mg).
A part of the n-BuOH extract (2.7 g) was fractioned on a Sephadex
5
5 × 10 cfu/mL. Sterile di-methyl sulfoxide (DMSO, Sigma-Aldrich)
(
(
LH20) column eluated with MeOH to give 19 fractions. Fraction 3
516.4 mg) was submitted to a semipreparative HPLC separation, using
and ciprofloxacin (1 mg/mL in DMSO) were used as negative and po-
sitive control, respectively. Plates were prepared in triplicate and in-
cubated at 37 °C for 24 h. The lowest concentration at which a color
change occurred (from dark purple to colorless) revealed the MIC value.
MeOH-H
compounds 1 (1 mg, t
2.2 mg, t = 14.1 min). Fraction 9 (44.6 mg) was purified by semi-
preparative HPLC using MeOH-H₂O (35/65) as mobile phase (Flow rate
.5 mL/min) to yield compound 9 (1.6 mg, t = 6 min). Fraction 17
15.9 mg) showed the formation of a precipitate which was filtered and
2
O (40/60) as mobile phase (flow rate 2.5 mL/min) to yield
R
= 8.8 min), 3 (1.4 mg, t = 11.2 min) and 6
R
(
R
Conflict of interest
2
(
R
The authors declared no conflict of interest.
Acknowledgments
washed with MeOH to afford compound 8 (4.4 mg)
.
3
.3.1. Centaureolide A (1)
2
5
Amorphous white solid; C20
H29NO
6
; [α]
D
+ 11.3 (c 0.1 MeOH); IR
This work has been performed in the frame of the bilateral project
Algeria-Italy “Exploitation of endemic plants of Algerian Sahara: che-
mical investigation and biological evaluation”, granted by the Italian
Ministry of Foreign Affairs and International Cooperation (MAECI). The
authors thank the MAECI General Management for the Cultural
Promotion and Cooperation, for its support.
KBr
−1
ν
max cm : 3430 (> OH), 2920 (> CH), 1775 (C=O), 1630 (C=C); for
1
13
H and C NMR (CD
3
OD, 600 MHz and 150 MHz, respectively) data see
−
Table 1; HR-ESIMS [M-H] m/z 378.1925 (calcd for C20
6
H28NO at m/z
378.1917).
3
.3.2. Centaureolide B (2)
2
5
Amorphous white solid; C20
H
29NO
6
; [α]
D
+ 9.1 (c 0.05 MeOH); IR
Appendix A. Supplementary data
KBr
−1
ν
max cm : 3435 (> OH), 2930 (> CH), 1775 (C=O), 1645 (C=C); for
1
13
H and C NMR (CD
Table 1; HR-ESIMS [M-H] m/z 378.1922 [M-H]
28NO at m/z 378.1917).
3
OD, 600 MHz and 150 MHz, respectively) data see
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fitote.2018.12.001.
−
−
(calcd for
C
20
H
6
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3.4. Acid treatment of centaureolide B (2)
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5
[
absolute configuration as L ([α]
D
− 80.2, c. 0.001, MeOH) [12,21].
3
3
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