Potent anti-platelet constituents from centaurea iberica

Article abstract of DOI:10.3390/molecules16032053

New naturally occurring nitrogenous compounds 1 and 2, along with a new dimeric lignan glucoside 3, have been isolated from the ethyl acetate soluble fraction of Centaurea iberica. Their structures have been elucidated through spectroscopic techniques. All the isolated compounds showed significant platelet aggregation inhibition.

Full text of DOI:10.3390/molecules16032053

Molecules 2011, 16, 2053-2064; doi:10.3390/molecules16032053
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Potent Anti-Platelet Constituents from Centaurea iberica
1
1
1
1,
Amna Nisar Khan , Itrat Fatima , Urooj Abdul Khaliq , Abdul Malik *, Ghulam Abbas
2
3
3
Miana , Zia-ur-Rehman Qureshi and Huma Rasheed
1
International Centre for Chemical and Biological Sciences, HEJ Research Institute of Chemistry,
University of Karachi, Karachi 75270, Pakistan
2
3
th
Riphah Institute of Pharmaceutical Sciences, Riphah International University, 7 Avenue,
G-7/4 Islamabad, Pakistan
International Centre for Chemical and Biological Sciences, Dr. Panjwani Centre for Molecular
Medicine and Drug Research, University of Karachi, Karachi 75270, Pakistan
*
Author to whom correspondence should be addressed; E-Mail: abdul.malik@iccs.edu;
Tel.: +92 21-2824926; Fax: +92 21-4819018.
Received: 3 January 2011; in revised form: 24 February 2011 / Accepted: 26 February 2011 /
Published: 28 February 2011
Abstract: New naturally occurring nitrogenous compounds 1 and 2, along with a new
dimeric lignan glucoside 3, have been isolated from the ethyl acetate soluble fraction of
Centaurea iberica. Their structures have been elucidated through spectroscopic techniques.
All the isolated compounds showed significant platelet aggregation inhibition.
Keywords: Centaurea iberica; nitrogenous compounds; dimeric lignan glucoside; anti
platelet activity
1
. Introduction
The genus Centaurea belongs to the family Asteraceae and comprises several hundred species
which are predominantly distributed around the Mediterranean area and Western Asia [1]. Many of
these species have been used in traditional medicine for the treatment of cancer [2], microbial
infections [3,4] and as stimulant, tonic [5,6], antadiabetic [7-9], diuretic [7] and antirheumatic [10,11].
Centaurea iberica is a herb commonly found in the northern areas of Pakistan. Previously, flavones,
steroid, fatty acids, volatile constituents, sesquiterpene lactones and other terpenoids have been
reported from this species [12-16]. Several interesting biological activities of this species have also

Molecules 2011, 16
2054
been reported, including the anti-inflammatory, wound healing and insulin secretion activities of the
plant extract [17,18]. The chemotaxanomic and ethnopharmacological importance of the genus
Centaurea prompted us to undertake further phytochemical studies on C. iberica. An ethanolic extract
of this plant showed strong toxicity in a brine shrimp lethality test [19]. Further fractionation revealed
strong toxicity in the ethyl acetate and n-butanol soluble sub-fractions. Pharmacological screening of
the ethyl acetate soluble sub-fraction revealed significant inhibition of platelet aggregation. A series of
chromatographic resolutions applied on this fraction have now resulted in the isolation and structural
elucidation of two new naturally occurring nitrogenous compounds, namely 3-methyl-2-benzyl-4-
quinazolone (1) and methyl-2-[(methylamino) carbonyl] benzoate (2), along with a new dimeric lignan
glucoside 3, respectively. Compounds 1–3 showed significant inhibition of platelet aggregation.
2
. Results and Discussion
The EtOAc soluble sub-fraction of the ethanolic extract of the whole plant of Centaurea iberica
was subjected to a series of column chromatographic techniques to obtain compounds 1-3 and their
structures established by UV, IR, MS and NMR spectroscopy.
Compound 1 was isolated as crystalline solid which gave a positive test for nitrogenous compounds
with Dragendorf’s reagent. The molecular formula C H N O was established by HREIMS showing
1
6
14
2
+
−1
−1
M peak at m/z 250.1106 (calc. 250.1115). In the IR spectrum the bands at 1,678 cm and 1585 cm
−1
were characteristic of the quinazolone system [20], while another band at 1,412 cm could be
attributed to a N-Me functionality. The UV spectrum showed absorption maxima at 227, 269, 273 and
3
11 nm, also characteristic of a quinazolone system [21]. The EIMS gave a distinct molecular ion peak
+
at m/z 250, while two other characteristic fragment ions at m/z 159 (M –CH
2
C
6
H
5
) and 91 (CH
2
C
6
H
5
)
1
3
revealed the presence of a benzylic moiety. The C-NMR (BB and DEPT) spectra of 1 corroborated
the presence of one methyl, one methylene, nine methine and five quaternary carbons. The downfield
signal at δ 164.0 could be attributed to the carbonyl carbon of the amide moiety while the signal at δ
2
8.2 could be assigned to the methyl group attached to nitrogen. The N-substituted aromatic carbon
resonated at δ 150.0 while the amide substituted aromatic carbon appeared at δ 121.8.
1
The H-NMR spectrum showed the characteristic signals of the aromatic protons of the quinazolone
system [δ 8.16 (1H, dd, J = 8.0, 1.5 Hz, H-5), 7.79 (1H, ddd, J = 8.0, 7.0, 1.5 Hz, H-7), 7.67 (1H, dd,
J = 1.5, 8.0 Hz, H-8) and 7.48 (1H, ddd, J = 8.0, 7.0, 1.5 Hz, H-6)]. The assignments were
1
1
corroborated with the help of H- H COSY and HMBC correlations (Table 1). The two homotopic
benzylic protons were observed as singlet at δ 4.00. The aromatic protons of the benzylic moiety were
2
observed between δ 7.35–7.30. Its position was confirmed by HMBC showing J correlation of the
benzylic protons at δ 4.00 with C-2 (δ 159.0). On the basis of these cumulative evidences, the structure
of compound 1 could be assigned as 3-methyl-2-benzyl-4-quinazolone (Figure 1). This is the first
report of the isolation of this compound from a natural source, following an earlier synthesis involving
selective methylation of glycosminine [20].

Molecules 2011, 16
2055
1
13
Table 1. H- (500 MHz) and C-NMR (125 MHz) data of 1 (CD
3
OD, J in Hz and δ in
ppm) and HMBC correlations.
1
13
1
Position
H
C
HMBC (correlation with H)
1
2
3
4
5
6
7
8
9
1
1
2
3
4
7
-
-
-
-
-
-
-
-
-
159.0
-
164.5
127.1
127.9
136.0
127.5
150.0
121.8
137.1
129.9
129.8
128.3
42.3
8.16 (dd, 8.0, 1.5)
7.48 (ddd, 8.0, 7.0, 1.5)
7.79 (ddd, 8.0, 7.0, 1.5)
7.76 (dd, 8.0, 1.5)
-
-
-
7.30 (m)
7.35 (m)
7.23 (m)
4.00 (s)
1.88 (s)
4, 6, 7, 9, 10
5, 7, 8
5, 6, 8, 9
6, 7, 9, 10
-
0
'
'/6'
'/5'
'
'
-
-
1',3',4',5'
1', 2', 4', 6'
2',3',5',6'
2,1',2',6'
2, 4
N-Me
28.2
Figure 1. Structure of compound 1.
O
5
3'
6'
1
0
3
4
2'
1'
4'
5
6
7
N
'
2
9
N
1
8
7'
+
Compound 2 was obtained as a white crystalline solid. Its HREIMS gave an M peak at m/z
93.0738 consistent with the molecular formula C10 (calc. 193.0744). The IR spectrum
1
H11NO
3
−1
−1
−1
revealed the presence of amide NH (3,400 cm ), an ester (1,695 cm ), amide carbonyl (1,675 cm ),
−1
−1
13
3
aromatic (1,500, 1,455 cm ) and N- CH (1,414 cm ) functionalities. The C-NMR spectra (BB and
DEPT, Table 2) showed 10 signals, comprising two methyl, four methine and four quaternary carbons.
The downfield signals at δ 171.4 and 169.7 were due to conjugated ester and amide moieties,
respectively. A singlet at δ 52.9 was characteristic of a methoxyl carbon, while another singlet at δ
2
4.8 could be ascribed to N-CH3. The di-substituted aromatic ring could be confirmed by downfield
1
signals at δ 141.7 and 131.5. The H-NMR spectrum (Table 2) showed a singlet at δ 4.88 for the amide
3
proton while two further singlets at δ 3.91 and 2.19 were due to O-CH and N-CH3, respectively. The
aromatic protons gave characteristic signals of 1, 2-di-substituted benzene ring with double doublets at
δ 8.43 (J = 8.5, 1.5 Hz, H-3) 8.40 (J = 8.0, 1.5 Hz, H-6) and doublet of double doublets at δ 7.52
(
J = 8.5, 7.0, 1.5 Hz, H-4) and 7.12 (J = 8.0, 7.0, 1.5 Hz, H-5). On the basis of these evidences along

Molecules 2011, 16
2056
with HMQC and HMBC correlations, the structure of compound 2 could be assigned as methyl-2-
(methylamino)carbonyl]benzoate (Figure 2). This is the first report of natural occurrence of this
[
compound following its earlier preparation via synthetic route [22].
Figure 2. Structure of compound 2.
6
O
1
2
2
'
5
1'
O
H
N
4
1''
2
''
3
O
1
13
Table 2. H- (500 MHz) and C-NMR (125 MHz) data of 2 (CD
3
OD, J in Hz and δ in
ppm) and HMBC correlations.
1
13
1
Position
H
C
HMBC (correlation with H)
1
2
3
4
5
6
-
-
141.7
131.3
122.0
-
-
8.43 (dd, 8.5, 1.5)
7.52 (ddd, 8.5, 7.0, 1.5) 135.2
7.12 (ddd, 8.0, 7.0, 1.5) 124.2
8.43 (dd, 8.0, 1.5)
-
3.91 (s)
-
2.19 (s)
1, 2, 4, 5, 1'
2, 3, 5, 6
1, 3, 4, 6
1, 2, 4, 5
-
1'
-
131.9
171.4
52.9
169.7
24.8
1
2
1
2
'
'
''
''
1''
Compound 3 was obtained as a yellow gum. Its molecular formula was established as C54
H
66
O
21 by
+
HRFABMS, which showed an [M+H] peak at m/z 1051.4174 (calc. 1051.4179). The molecular
formula was further confirmed through elemental analysis and low resolution ESI-MS showing
+
−1
[
M+H] peak at m/z 1,051. The IR spectrum showed hydroxyl group (3,400 cm ), a γ-lactone
−1
−1
(
1,760 cm ) and aromatic (1,590 and 1,514 cm ) functionalities. The UV absorption bands at 228 and
1
2
80 nm were assignable to the multiple substituted aromatic rings. The H-NMR spectrum (Table 3)
was consistent with those of trans-dibenzylbutyro lactone derivatives [23]. The benzylic protons at δ
C-7 with H-8 and H-8' give a complex multiplet at δ 2.48–2.67, while the other benzylic protons (H
') are more de-shielded due to the proximity of the lactone carbonyl and appear as double doublets at
δ 2.98 and 2.80. The two protons at C-9 resonated characteristically as double doublets, at δ 4.18 and
.93. The rest of the spectrum revealed the presence of six aromatic protons and three methoxyl
2
-
7
3
groups. These were consistent with the presence of a guaiacyl ring and a vertaryl ring in the molecule.
It was also supported by EIMS which showed prominent fragments at m/z 372, 151 and 137
corresponding to benzyl fragments, one of these being substituted with one hydroxy and one methoxy
groups and the other containing the remaining methoxy groups.

Molecules 2011, 16
2057
1
13
Table 3. H- (500 MHz) and C-NMR (125 MHz) data of 3 (CD
3
OD, J in Hz and δ in
ppm) and HMBC correlations.
1
13
1
Positions
H
C
HMBC (correlation with H)
1
2
3
4
5
6
7
8
9
-
132.7
114.8
150.7
149.2
113.1
122.1
38.9
-
6.74 (d, 1.5)
-
-
6.81 (d, 8.0)
6.59 (br s)
2.53 (m)
2.48 (m)
4.18 (dd, 7.5, 9.0)
1, 3, 4, 6, 7
-
-
1, 3, 4, 6
1, 2, 4, 5, 7
8', 1, 2, 6, 8, 9
7', 8', 9', 1, 7, 9
8', 9', 7, 8
42.5
72.9
3
-
.93 (dd, 7.5, 9.0)
1
2
3
4
5
6
7
'
'
'
'
'
'
'
134.3
113.6
150.4
146.9
117.8
123.0
35.4
-
6.61 (d, 1.5)
-
-
7.03 (d, 8.5)
6.63 (dd, 8.0, 1.5)
2.98 (dd, 5.0, 13.5)
1', 3', 4', 6', 7'
-
-
1', 3', 4', 6'
1', 2', 4', 5', 7'
1', 2', 6', 8', 9', 8
2
.80 (dd, 7.5, 13.5)
8
9
1
2
3
4
5
6
'
'
2.67 (m)
-
47.6
181.3
102.9
74.9
78.1
71.3
77.8
62.5
1', 7', 9', 7, 8, 9
-
''
''
''
''
''
''
4.88 (d, 7.5)
3.47 (m)
3.93 (m)
3.38 (m)
3.46 (m)
3.66 (br d, 12.5)
4', 2'', 3''
1'', 3'', 4''
1'', 2'', 4'', 5''
2'', 3'', 5'', 6''
3'', 4'', 6''
4'', 5''
3
.84 (br d, 12.5)
3-OMe
4-OMe
3'-OMe
3.79 (s)
3.79 (s)
3.74 (s)
56.5
56.5
56.7
3
4
3'
The presence of a hexose moiety was evident by an anomeric proton at δ 4.88 (J = 7.5 Hz). Its
larger coupling constant was in conformity to the β-linkage of the sugar moiety. Other oxymethine and
oxymethylene protons are shown in Table 3. Acid hydrolysis of 3 provided D-glucose, which could be
identified through the sign of its optical rotation and comparison of retention time of its TMS ether
with a standard in GC. The HMBC correlation of anomeric proton at δ 4.88 with C-4 (δ 149.2)
confirmed the point of attachment of the glucose moiety. This could be authenticated by close
1
13
similarity of H- and C-NMR data of 3 with those of arctiin [24]. Insofar however, as the two
compounds differ in specific rotations and the molecule has twice as many carbon atoms as arctin,
compound 3 must be a symmetrical dimer involving two molecules of arctin. Concluding evidence was
+
provided by acetylation of 3, which provided a tetraacetyl derivative showing a [M+H] peak in
1
HRFABMS at m/z 1,219.4597 (calc. 1,219.4604). The careful comparison of its H-NMR spectrum

Molecules 2011, 16
2058
with that of 3 revealed downfield shifts of H-2'' and H-3'' due to the acetylation of both the hydroxyl
groups at C-2'' and C-3'', respectively. On the other hand, H-4'' remained unaffected providing
evidence that the hydroxyl groups at C-4'' of the sugar moieties of both the monomers are involved in
ethereal band formation. Conclusive evidence was provided by dissolving compound 3 in D O-CD OD
2
3
+
and repeating the ESI-MS which now gave an [M+H] peak at m/z 1057 due to deuterium exchange of
six acidic alcoholic sugar protons [25]. Hence the structure of compound 3 could be assigned as
illustrated in Figure 3.
Figure 3. Structure of compound 3.
MeO
4
MeO
3
5
O
O
O
2
6
MeO
6''
O
4''
1
7
O
5'
O
O
O
O
4'
O
O
9
6'
8
O
O
O
1''
8'
9'
3'
1'
OMe
7'
O
2'
OMe
OMe
The results of the antiplatelet activity demonstrated that treatment of PRP with arachidonic acid
0.5–1.7 mM) showed a concentration-dependent increase in platelet aggregation. The maximum
(
aggregation was obtained with 1.73 mM of arachidonic acid. We found that the effect of arachidonic
acid was inhibited by pre-treatment of PRP with compounds 1-3. All of these compounds inhibited
human platelet aggregation in a concentration dependent manner (compound 1 = 100, 150 and 200 μM;
compound 2 = 50, 75 and 100 μM and compound 3 = 100, 200 and 400 μM) (Figure 4). The IC50
values are illustrated in Table 4. The results indicated that all the three compounds were active against
arachidonic acid induced platelet aggregation as compared to the effect of aspirin, which inhibits TXA
formation. TXA is a potent mediator of platelet aggregation released from platelets when incubated
with arachidonic acid. The underlying mechanism by which the active metabolites of arachidonic acid
AA), i.e., thromboxane A and/or prostaglandin H (TXA /PGH ) induce platelet aggregation is still
2
2
(
2
2
2
2
unclear. Several reports have suggested that AA-stimulated aggregation is mediated by secreted ADP,
whereas other studies have proposed that this response is ADP-independent. Arachidonic acid is the
precursor of prostanoids which liberates from lipid membrane of platelets by the action of
2
phospholipase A2. AA is processed in two stages by cyclooxygenase to produce prostaglandin (PG) H .
This process is aspirin-sensitive, as the drug irreversibly acetylates and inhibits cyclooxygenase that
shows the basis for the effective use of aspirin as antithrombotic agent, which itself underscores its
involvement in arterial thrombosis. Thromboxane synthase then consumes a proportion of PGH
generate thromboxane (TXA ) that activates platelet aggregation [26] (Figure 5).
2
to
2

Molecules 2011, 16
Figure 4. Tracings from the representative experiments showing the effect of various
2059
inhibitors on arachidonic acid induced platelet aggregation. (A = compound 1;
B = compound 2; C = compound 3 and D = aspirin showing inhibition of arachidonic acid
induced platelet aggregation in a dose-dependent manner. Control = arachidonic acid
(
1.73 mM), N = 8–10.
Figure 5. Schematic representation of arachidonic acid metabolism in platelets through
cyclooxygenase that catalyzes the synthesis of prostaglandins, thromboxane A
prostacyclins.
2
and

Molecules 2011, 16
2060
Table 4. The effects of compounds 1-3 on arachidonic acid (1.73 mM) induced platelet
aggregation.
Inhibitors
Mean IC50 M  SEM
160  3.5
Compound 1
Compound 2
Compound 3
Aspirin
75 ± 2.1
45 ± 2.8
150  4.4
Data is mean ± SEM (n = 8–10) and is indicated as half-maximal effect (IC50) of the inhibitors.
It is proposed that these compounds may produce anti-platelet effects due to the inhibition of
cycloxygenase enzyme responsible for the synthesis of thromboxane A like aspirin. Arachidonic acid
2
was used as potent platelet aggregating agent which is the substrate of cycloxygenase enzyme that
2
synthesizes thromboxane A in the platelets. It is the most potent mediator of platelet aggregation that
releases from platelets and acts on its receptor on the surface of the platelets in an autocrine fashion
and mediates platelet aggregation [27]. As we compared the effects of these compounds with aspirin
that has the same mechanism in inhibiting COX enzyme followed by the inhibition of TXA , we
2
preferred arachidonic acid in this study as it has a major role in platelet. From the present in vitro
studies it can be concluded that compounds 1-3 have potential for future development as anti platelet
drugs. However, in vivo studies could not be carried out at this time due to paucity of materials.
3. Experimental
3.1. General
Optical rotations were measured on a JASCO DIP-360 polarimeter. IR spectra were recorded on a
60 Shimadzu spectrometer. The UV spectra were recorded on a Hitachi UV-3200 spectrometer. The
4
HREIMS were recorded on JEOL JMX-HX 110 mass spectrometer while HRFABMS was recorded on
JEOL JMS-DA-5000 mass spectrometer. ESI mass spectrum was obtained on QSTAR XL
1
13
spectrometer. The H- and C-NMR, HMQC and HMBC spectra were recorded on Bruker
1
13
spectrometer operating at 500 MHz for H– and 125 MHz for C-NMR, respectively. The chemical
shift values are reported ppm (δ) units and J are in Hz. The parameters for 1D and 2D NMR spectra
were similar those reported by us previously [28]. The elemental analysis was performed on a Perkin-
Elmer elemental analyzer, model 240. Aluminum sheets pre-coated with silica gel 60 F254 (20 × 20 cm,
0
.2 mm thick, E-Merck, Darmstadt, Germany) were used for thin layer chromatography (TLC) and
silica gel (230–400 mesh, E-Merck, Darmstadt, Germany) was used for column chromatography. The
purity of the compounds was routinely checked by TLC using HPTLC precoated glass plates of silica
gel 60 F254 (20 × 10 cm., E. Merck, Darmstadt, Germany). Platelet aggregation was monitored on dual
channel Lumi aggregometer (Model 400, Chronolog Corporation, Chicago, IL, USA). EtOH, AcOEt,
n-hexane, n-butanol, Arachidonic acid and sodium citrate were purchased from Sigma Chemical Co.
(
St. Louis, MO, USA). All other chemicals used were of the highest purity grade. Arachidonic acid
was reconstituted in ethanol and 0.2% w/v aqueous solution of sodium carbonate. The contents were
then vortexed and transferred under nitrogen to screw capped vial to avoid auto-oxidation.

Molecules 2011, 16
.2. Plant Material
The whole plant material of Centaurea iberica Trev. was collected from Islamabad in April, 2007
2061
3
and identified by Prof. S. Iftikhar Hussain Shah, Department of Pharmacognosy, Riphah International
University, Islamabad. A voucher specimen has been deposited in the herbarium of Plant Sciences,
Quaid-i-Azam University, Islamabad (voucher specimen no. ISL 49920).
3
.3. Extraction and Isolation
The shade dried whole plant material (20 kg) was extracted with EtOH (3 × 50 L) at room
temperature. The combined ethanolic extract was evaporated under reduced pressure to obtain a thick
gummy residue (300 g) which was suspended in water and successively extracted with n-hexane, ethyl
acetate and n-butanol. The ethyl acetate soluble fraction (100 g) was subjected to column
chromatography eluting with mixtures of n-hexane, EtOAc and MeOH in increasing order of polarity
to obtain six fractions (A-F). The fraction B obtained from n-hexane-EtOAc (8:2) was
rechromatographed and eluted with n-hexane-EtOAc (7:3) to obtain compounds 1 (10 mg) and 2
(
(
14 mg) from the head and tail fractions, respectively. The fraction F obtained from EtOAc-MeOH
9:1) was rechromatographed and eluted with mixtures of n-hexane, EtOAc and MeOH in increasing
order of polarity. The fraction which eluted with EtOAc-MeOH (9:1) was a semi pure compound
which was purified by further chromatography eluting with EtOAc-MeOH (9.2:0.8) to furnish
compound 3 (20 mg).
3
-Methyl-2-benzyl-4-quinazolone (1): Crystalline solid. UV (MeOH) λmax (log εmax): 227 (3.25), 269
−1
(
(
3.01), 273 (3.42), 311 (2.78) nm. IR (KBr): γ = 1,678, 1,585, 1,412 cm . EIMS m/z (rel. int.): 250
1
20), 236 (60.1), 159 (25), 91 (100). HREIMS: m/z 250.1106 (calcd. 250.1115 for C16
H
14
N
2
O). H-
1
3
and C-NMR: see Table 1.
Methyl-2-[(methylamino)carbonyl]benzoate (2): White crystalline solid. UV (MeOH) λmax (log εmax):
−1
2
08 (3.12), 231 (3.13), 169 (3.28) nm. IR (KBr): γ = 3,400, 1,695, 1,675, 1,500, 1,455, 1,414 cm .
EIMS m/z (rel. int.): 193 (20), 179 (45), 120 (100). HREIMS: m/z 193.0738 (calcd. 193.0744 for
1
13
10 3
C H11NO ). H- and C-NMR: see Table 2.
(
3R,4R)-4-(3,4-dimethoxybenzyl)-3-(4-{[5-{[6-(4-{[(3R,4R)-4-(3,4-dimethoxybenzyl)-2-oxotetrahydro-
-furanyl]methyl}-2-methoxyphenoxy)-4,5-dihydroxy-2-(hydroxylmethyl)tetrahydro-2H-pyran-3-yl]
oxy}-3,4-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]oxy}-3-methoxybenzyl)dihydro-2-
3H)-furanone (3): Yellow gum. UV (MeOH) λ (log εmax): 228 (4.12), 280 (4.08) nm. IR (KBr):
3
(
max
−1
γ = 3,400, 1,760, 1,590, 1,514 cm . EIMS m/z (rel. int.): 372 (35), 151 (69), 137 (100). HRFABMS:
m/z 1051.4174 (calcd. 1051.4179 for C54 21). ESIMS: m/z 1051 and m/z 1057 (after dissolving
OD). H- and C-NMR: see Table 3. Anal. Calcd. for C54 21 : C, 61.71,
H, 6.28, O,32.00 ; Found : C, 61.67, H, 6.23, O, 31. 89.
67
H O
1
13
compound 3 in D
2
O-CD
3
66
H O

Molecules 2011, 16
2062
3
.4. Acid Hydrolysis of Compound 3
A solution of compound 3 (4.2 mg) in MeOH (5 mL) containing 1N HCl (2 mL) was refluxed for
h, concentrated under reduced pressure, diluted with H O and extracted with EtOAc. The organic
4
2
phase provided the aglycone as colorless gummy solid which could be identified as arctigenin through
comparison of spectral data with those reported in literature [24]. The aqueous phase was concentrated
23
to obtain the glycone which could be identified as D-glucose by the sign of its optical rotation ([α]D
+
5
1.8 (c = 0.02, MeOH) and comparison of retention time of its TMS ether with that of standard in gas
chromatography. The preparation of TMS ether and conditions of GC were same as reported
previously in literature [24].
3
.5. Preparation of Human Platelets
Blood was drawn by vein-puncture from healthy human subjects who had not taken any medicine
for at least one week. Each sample was mixed with 3.8% (w/v) sodium citrate solution (9:1) and
centrifuged at 260 g for 15 min at 20 °C to obtain platelet rich plasma (PRP). Platelet count was
determined by phase contrast microscopy and all aggregation studies were carried out at 37 °C with
8
−1
PRP having platelet count between 2.5 and 3.0 × 10 mL of plasma [27]. Platelet poor plasma (PPP)
was obtained by centrifuging blood at 400 g for 5 min and was used as a blank source in the
aggregometer. All experiments were performed within 2 h of PRP preparation.
3.6. Measurement of Platelet Aggregation
Aggregation was monitored using Dual-channel Lumi-aggregometer using 0.45 mL aliquots of PRP
in a glass cuvette. The final volume was made up to 0.5 mL with the test drug dissolved either in
normal saline or appropriate vehicle known to be devoid of any effect on aggregation. Dual-channel
Lumi-aggregometer works on the principle of light transmission. The more platelet are activated the
greater aggregation is observed after adding the stimulating agent. The test compounds were dissolved
in DMSO in such a way that the final concentration in cuvette was less than 1% as DMSO itself has an
inhibitory effect on platelets. Aggregation was induced with arachidonic acid (1.73 mM) and the anti
platelet activity of different compounds were studied by pretreatment of PRP with various
concentrations of the inhibitors for 1 min followed by the addition of arachidonic acid. The resulting
aggregation was recorded for 5 min after challenge by the change in light transmission as a function of
time. Once the anti platelet activity of various inhibitors against agonists was established, dose
response curves were constructed to calculate the IC50 values of the agonists and compounds. Aspirin,
a potent anti-platelet agent was taken as a reference drug.
3
.7. Data Analysis
IC50 = Concentration (μM) producing 50% inhibition of platelet aggregation (control response taken
as 100%). The IC were calculated as means ± SEM of 5–6 independent experiments. Differences
5
0
between control and test measurements were assessed by student’s t-test.

Molecules 2011, 16
. Conclusions
In conclusion, the new naturally occurring nitrogenous compounds 1 and 2, along with a new
2063
4
dimeric lignan glucoside 3, have been isolated from C. iberica and their structures have been
elucidated through spectroscopic techniques. All the isolated compounds showed significant inhibition
of platelet aggregation.
Acknowledgements
The authors are grateful to Higher Education Commission of Pakistan for research funding.
References
1
.
Mabberley, D. The Plant-Book, 2nd ed.; Cambridge University Press: Cambridge, UK, 1997;
p. 138.
2
3
.
.
Grieve, M. A Modern Herbal; Dover Publications, Inc.: New York, NY, USA, 1971.
Kumarasamy, Y.; Nahar, L.; Cox, P.J.; Dinan, L.N.; Ferguson, F.R.; Finnie, D.A.; Jaspars, M.;
Sarker, S.D. Biological activities of lignans from Centaurea scabiosa. Pharm. Biol. 2003, 41,
2
03–206.
4
.
Panagouleas, C.; Skaltsa, H.; Lazari, D.; Skaltsounis, A.L.; Sokovie, M. Antifungal activity of
secondary metabolites of Centaurea raphanina ssp. Mixta, growing wild in Greece. Pharm. Biol.
2
003, 41, 266–270.
5
6
.
.
Bitar, I.-E. Mofradat Al-Adwiah Wa Al-Agzia; Al-Zharia Press: Cairo, Egypt, 1980; p. 148.
Wat, J.M.; Breyer-Brandwijik, M.G. The Medicinal and Poisonous Plants of Southern and
Eastern Africa, 2nd ed.; E. & S. Livingstone Ltd.: Edinburgh, London, UK, 1962; p. 210.
Monya, M.; Racz, G. Content of flavonosides in some species of the genus Centaurea L.I.
Chromatographic studies. Plant Med. Phytother. 1974, 8, 126.
7.
8.
9.
1
1
Masso, J.L.; Bertran, M.N.; Adzet, T. Contribution to the chemical and pharmacologic study of
some species of Centaurea (Compositae). Plant Med. Phytother. 1979, 13, 41–45.
Kaij-A-Kamb, M.; Amoros, M.; Girrel, L. Chemistry and biological activity of the genus
Centaurea. Pharma. Acta Helv. 1992, 67, 178–188.
0. Gonzalez, A.G.; Bermejo, J.; Caberar, I.; Galido, A.; Masenet, G.M. Sesquiterpene lactones from
Centaurea alba and C. conifera. Ann. Quim. 1977, 73, 86.
1. Negrette, R.E.; Latorre, I.; Backhouse, N.; Delpote, C. Guaianolides from Centaurea scoparia.
Plant Med. Phytother. 1988, 53, 503.
1
2. Ahmed, N.; Bibi, R. Chemical investigation of Centaurea iberica. Fitoterapia 1979, 50, 199–200.
3. Tekeli, Y.; Sezgin, M.; Aktumsek, A.; Guler, G.O.; Sanda, M.A. Fatty acid composition of six
Centaurea species growing in Konya, Turkey. Nat. Prod. Res. 2010, 24, 1883–1889.
4. Senatore, F.; Arnold, N.A.; Bruno, M. Volatile components of Centaurea eryngioides Lam. and
Centaurea iberica Trev. Var. hermonis Boiss. Lam., two Asteraceae growing wild in Lebanon
Nat. Prod. Res. 2005, 19, 749–754.
1
1
1
5. Sham’yanov, D.; Akhmedov, U.A.; Saidkhodzhaev, A.I. Sesquiterpene lactones and other
components of Centaurea iberica. Chem. Nat. Compd. 1998, 34, 339–340.

Molecules 2011, 16
2064
1
1
1
6 Dumlu, M.U.; Gürkan, E. A new active compound from Centaurea species. Z. Naturforsch. C.
006, 61, 44–46.
2
7. Koca, U.; Süntar, I.P.; Keles, H.; Yesilada, E.; Akkol, E.K. In vivo anti-inflammatory and wound
healing activities of Centaurea iberica Trev. ex Spreng. J. Ethnopharmacol. 2009, 126, 551–556.
8. Hussain, Z.; Waheed, A.; Qureshi, R.A.; Burdi, D.K.; Verspohl, E.J.; Khan, N.; Hasan, M. The
effect of medicinal plants of Islamabad and Murree region of Pakistan on insulin secretion from
INS-1 cells. Phytother. Res. 2004, 18, 73-77.
1
9. Meyer, B.N.; Ferrigni, N.R.; Putman, J.E.; Jacobson, L.B.; Nichols, D.E.; McLaughlin, J.L. Brine
shrimp: a convenient general bioassay for active plants constituents. Planta Med. 1982, 45, 31–34.
0. Pakrashi, S.C.; Bhattacharyya, J. Studies on indian medicinal plants. Tetrahedron 1968, 24, 1–5.
2. Laumer, J.Y.S.; Frerot, E.; Herrmann, A. Controlled release of perfumery alcohols by
neighboring-group participation. Comparison of the rate constants for the alkaline hydrolysis of 2-
Acyl-,2-(hydroxymethyl)-, and 2-carbamoylbenzoates. Helv. Chim. Acta 2003, 86, 2871–2899.
3. Juraj, H.; Milos, B.; Antonin, T. Plant substances XLV. The structure of yatein. Determination of
the positions and configurations of benzyl groups in lignans of the 2,3-dibenzylbutyrolactone
type. Collect. Czech Chem. Commun. 1982, 47, 644–663.
2
2
2
2
2
4. Maiada, M.A.R.; Paul, M.D.; David, E.J. Lignan of Forsythia intermedia. Phytochemistry 1990,
2
9, 1971–1980.
5. Ricci, A.; Fiorentino, A.; Piccolella, S.; Golino, A.; Pepi, F.; D'Abrosca, B.; Letizia, M.; Monaco,
P. Furofuranic glycosylated lignans: A gas-phase ion chemistry investigation by tandem mass
spectrometry. Rapid Commun. Mass. Spectrom. 2008, 22, 3382–3392.
2
6. Farndale, R.W.; Hargreaves, P.G.; Dietrich, J.L.; Keogh, R.J. Measurement of platelet arachidonic
acid metabolism. Methods Mol. Biol. 2004, 272, 121–133.
2
7. Rasheed, H.; Saeed, S.A. Involvement of thromboxane A2 and tyrosine kinase in the synergistic
interaction of platelet activating factor and calcium ionophore A23187 in human platelet
aggregation. Exp. Mol. Med. 2004, 114, 376–380.
2
8. Ahmad, Z.; Mehmood, S.; Fatima, I.; Malik, A.; Ifzal, R.; Afza, N.; Iqbal, L.; Latif, M.; Nizami,
T.A. Structural determination of salsolins A and B, new antioxidant polyoxygenated triterpenes
from Salsola baryosma, by 1D and 2 D NMR spectroscopy. Magn. Reson. Chem. 2008, 46,
9
4–98.
Sample Availability: Samples of compounds 1–3 are available from the authors.
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2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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