Pl. Syst. Evol.

261: 217–228 (2006)

DOI 10.1007/s00606-006-0448-3

A multivariate statistical approach to Centaurea classification using

essential oil composition data of some species from Turkey
G. Flamini1, M. Tebano1, P. L. Cioni1, Y. Bagci2, H. Dural2, K. Ertugrul2, T. Uysal2,
and A. Savran3
1
Dipartimento di Chimica Bioorganica e Biofarmacia, Pisa, Italy
2
Department of Biology, Faculty of Science and Art, Selcuk University, Konya, Turkey
3
Department of Biology, Faculty of Science and Art, Nigde University, Nigde, Turkey

Received December 6, 2005; accepted April 24, 2006

Published online: July 20, 2006

Springer-Verlag 2006

Abstract. The composition of the essential oils of
ten Centaurea species from Turkey, Centaurea
aladaghensis, C. antiochia var. prealta, C. antitauri,
C. babylonica, C. balsamita, C. cheirolepidoides,
C. deflexa, C. iconiensis, C. lanigera, C. ptosimop-
appoides have been studied. Multivariate statistical
analyses (Principal Component Analysis, Multidi-
mensional Scaling, Hierarchical Cluster Analysis)
applied to GC-MS data, seem to be very useful to
investigate and establish the natural taxonomic
delimitation of this very difficult genus. The
groupings resulted independent from the ecological
similarities (i.e. plants that live in the same habitats
or share similar morphological characteristics), so
it seems that the environment has no influence on
the biosynthesis of the volatiles of these plants.
Key words: Centaurea aladaghensis, C. antiochia
var. prealta, C. antitauri, C. babylonica,
C. balsamita, C. cheirolepidoides, C. deflexa,
C. iconiensis, C. lanigera, C. ptosimopappoides,
Essential oil composition, Turkey.
Introduction
In Turkey, the genus Centaurea (Asteraceae) is
represented by a very large number of species,
distributed in particular the Southwest, Central
and East of the country. Taxonomically this
taxon is very difficult and needs further studies,
mainly using modern cytological and chemical
techniques. The unnatural circumscription of
Centaurea is a very old problem (Wagenitz
1975) that arises from the large morphological,
karyological and palynological diversity (Bre-
mer 1994, Susanna et al. 1995, Wagenitz and
Hellwig 1996, Garcia-Jacas et al. 2001).
This paper deals about the essential oil
composition of ten endemic Centaurea species
from Turkey: C. aladaghensis, C. antiochia var.
prealta, C. antitauri, C. babylonica, C. balsamita,
C. cheirolepidoides, C. deflexa, C. iconiensis,
C. lanigera, C. ptosimopappoides, which have
never previously been isolated. We have chosen
both taxa growing in the same habitats, with
similar ecological needs, and taxa which show
morphological similarities to evaluate if the
pedoclimatic conditions could influence the
essential oil composition leading to a chemical
convergence.
Furthermore, in order to evaluate whether
the identified essential oil constituents could be
useful in defining the natural delimitation of this
218 G. Flamini et al.: Centaurea classification using chemometric data
genus, the constituents of all the essential
oils were subjected to multivariate statistical
analysis by means of Principal Component
Analysis (PCA), Multidimensional Scaling
(MDS) and Hierarchical Cluster Analysis
(HCA).
PCA is a well-known method where the
principal components (PCs) are calculated in
order to keep most of the information present
in the original data set in the least possible
number of new variables, usually two or three.
The PCs can be plotted for visual inspections
of the data to point out patterns hidden in the
dataset (Marengo et al. 1991).
Multidimensional scaling (MDS) treats the
dissimilarity matrix as a distance matrix. It
provides a visual representation of the pattern
of proximities (i.e. similarities or distances)
among a set of objects. MDS plots the objects
on a map such that those that are very similar to
each other are placed near each other on the
map, and those that are perceived different from
each other are placed far away from each other
on the map. As with PCA, the first few dimen-
sions are the most significant. Indeed, the results
of PCA and classical scaling are equivalent
under certain circumstances (Chatfield and
Collins 1980).
HCA is a method in which samples are
considered as lying in a n-dimensional space
and distances between samples are calculated
joining the objects with an agglomerative
procedure (Everitt 1980, Davis 1986).
Many papers about the secondary metabo-
lites of Centaurea species can be found in the
literature, but studies on the volatile constitu-
ents are, at present, limited to few species. These
latter investigations refer to C. depressa from
Iran (Esmaeili et al. 2005), C. eryngioides and
C. iberica from Lebanon (Senatore et al. 2005),
C. cineraria ssp. umbrosa from Italy (Senatore
et al. 2003), C. sessilis and C. armena (Yayli et al.
2005), C. dichroa (Altintas et al. 2004), C. mu-
cronifera and C. chrysantha (Dural et al. 2003),
C. pseudoscabiosa subsp. pseudoscabiosa and
C. hadimensis (Flamini et al. 2002) and C. kots-
chyi var. kotschyi and C. kotschyi var. decum-
bens from Turkey, (Ertugrul et al. 2003) C. pelia,
C. thessala subsp. drakiensis, C. zuccariniana,
C. raphanina subsp. mixta, C. spruneri from
Greece, (Lazari et al. 1999, 2000) and C.
calcitrapa and C. solstitialis from U.S.A. (But-
tery et al. 1986; Binder et al. 1990a, b). Other
earlier studies showed only partially character-
ized essential oils of C. calcitrapa, C. gloriosa
and C. moschata. (Karawya et al. 1975, Saleh
et al. 1981, Kustrak and Radic 1985).
No previous studies about the essential oils
from these species are present in the literature.
Only C. ptosimopappoides and C. babylonica
were previously investigated for its non-vola-
tile secondary metabolites. Five triterpenes
were isolated from the roots of the former,
together with scopoletin, 7-oxositosterol, stig-
masterol, a-amyrin, cynaropicrin, and 11,13-
dihydro-desacylcynaropycrin from the aerial
parts (Öksuz and Serin 1997). From the latter
six guaianolides were identified (Bruno et al.
2005). Another derivative identified for the
first time in the genus was syringin (Öksuz and
Ulubelen 1998).
C. aladaghensis Wagenitz (sect. Cynaroides)
is a perennial plant with at least 40 cm erect
stem, sparsely hairy leaves, triangular append-
age with spine and purple-colored flowers. This
plant is an endemic species for Turkey.
C. antiochia Bioss. var. praealta (Boiss. &
Bal.) Wagenitz (sect. Acrocentron) (Syn: Cen-
taurea praealta Boiss. & Bal.) is a perennial
plant with up to 100 cm erect stem, leaves with
tomentose to subglabrous hairs, small append-
ages with spine nearly orbicular appendage,
and blackish-purple-colored flowers. This plant
is an endemic species for Turkey.
C. antitauri Hayek (sect. Pseudophaeopap-
pus) (Syn: Phaeopappus rupestris Boiss. &
Hausskn., Tomanthea rupestris (Boiss. & Haus-
skn.) Takht.) is a perennial plant with up to 45
cm erect stem, leaves with septate, small, trian-
gular appendage and yellow-colored flowers.
This plant is an endemic species for Turkey.
C. babylonica (L.) L. (sect. Microlophus)
(Syn: Serratula babylonica L., Microlophus
babylonicus (L.) Sojak.) is a biennial plant
with up to 300 cm erect stem with numerous
capitula, grey-tomentose leaves, coriaceous
G. Flamini et al.: Centaurea classification using chemometric data 219

phyllaries, without appendages and yellow-
colored flowers.
C. balsamita Lam. (sect. Stizolophus ) (Syn:
Stizolophus balsamitaefolius Cass.) is an annual
species, up to 120 cm long, with scabrous
leaves, ovoid involucre, cartilaginous append-
ages, and yellow-colored flowers.
C. cheirolepidoides Wagenitz (sect. Pseudo-
seridia) is a perennial plant with about 40 cm
long erect stem, leaves with grey-tomentose
hairs, very small, triangular appendage, and
yellow-colored flowers. This plants is an
endemic species for Turkey.
C. deflexa Wagenitz (sect. Cheirolepis)
(Syn: Phaeopappus declinatus Boiss., Centau-
rea nivea (Bornm.) Wagenitz. var. declinata
(Boiss.) Wagenitz) is a perennial plant with
5–30 cm decumbent stem, leaves with densely
grey-tomentose hairs, nearly orbicular append-
age, and yellow-colored flowers. This plant is
an endemic species for Turkey.
C. iconiensis Hub.-Mor. (sect. Centaurea) is
a perennial plant with 60–80 cm long erect
stem, glabrous leaves, multiseriate and subco-
riaceous phyllaries, and gold yellow-colored
scarcely radiant flowers. This plant is an
endemic species for Turkey.
C. lanigera DC. (sect. Cyanus) (Syn: Cy-
anus lanigerus (DC.) Holub) is a perennial
plant with 5–10 cm long several prostrate or
ascending stem, loosely villous or slightly
tomentose leaves, appendages with numerous
cilia, white-colored radiant flowers. This plant
is an endemic species for Turkey.
C. ptosimopappoides Wagenitz (sect. Ptosi-
mopappus) is a perennial subschrub with about
35 cm, erect or ascending stem, firm, glabrous
leaves, appendages with a short mucro, and
yellow-colored flowers. This plant is an en-
demic species for Turkey.
Materials and methods
The capitula of Centaurea cheirolepidoides,
C. balsamita, C. aladaghensis, C. antitauri, C. lanigera,
C. deflexa, C. iconiensis, C. antiochia var. praealta,

Table 1. Origin and voucher specimens of the plant material

Species Voucher
C. aladaghensis Wagenitz Nigde: Camardi; the slopes of Mazmili dagi, under Pinus nigra forest, 1600–
1700 m., 15.VI.2002, Bagci 2956.
C. antiochia Boiss. var. Icel: Silifke, 8 km southern of Uzuncaburc, under Pinus forest, 900 m,
praealta (Boiss. & Bal.) 29.VI.2003, Ertugrul 2853-a.
Wagenitz
C. antitauri Hayek Nigde:Camardi; the slopes of Mazmili dagi, under Pinus nigra forest, 1600–
1700 m., 15.VI.2002, Bagci 2955.
C. babylonica (L.) L. Adana, Pozanti; the roadsides of Hamidiye and Buyuk Sofulu, 15.VII.2002,
Bagci 2957 & Savran.
C. balsamita Lam. Konya: the campus area of the Selcuk University, steppe, 1050 m, 15.VII.2001,
Bagci 2892.
C. cheirolepidoides Konya: Hadim Gevne vadisi, the west of Goztasi, in open area of Abies and
Wagenitz Cedrus forest, 1500 m, 36
520 1900 N, 32
4200 5700 E, 08.VII.2000, Ertugrul
2268.
C. deflexa Wagenitz Konya, Taskent, 25 km from Taskent to Ermenek, Feslikan yaylasi, steppe,
31.VII.2000, 36
470 5700 N, 32
370 1900 E, Ertugrul 2315.
C. iconiensis Hub.-Mor. Konya: 20 km from Seydisehir to Bozkir, roadside, 14. VII. 2001,
37
200 7900 N ; 32
0560 4900 E, Ertugrul 2480.
C. lanigera DC. Nigde: Aladag; Gokgol, Kargedigi mevki, 2900–3000 m., rocky places,
06.VII.2002, Bagci 2961& Savran.
C. ptosimopappoides Adana: Aladag; Terlik tepe, Atolugu akari, 1500–1600 m., 15.VI.2002, Bagci
Wagenitz 2951.
220 G. Flamini et al.: Centaurea classification using chemometric data
C. ptosimopappoides, C. babylonica were collected in
different localities of Turkey; voucher specimens of
these plants are deposited in KNYA Selcuk Univer-
sity, Science and Art Faculty, The Herbarium of
Biology Department (Table 1).
The plant material (150 g each), constituted
only of fresh capitula, obtained by collective
sampling, was separately hydrodistilled in a Cle-
venger-like apparatus for two hours. The oil was
than aspirated from the water surface by mean of a
syringe.
The GC analyses were accomplished with a
HP-5890 Series II instrument equipped with HP-
WAX and HP-5 capillary columns (30 m ·
0.25 mm, 0.25 lm film thickness), working with
the following temperature program: 60C for
10 min, ramp of 5C/min up to 220C; injector
and detector temperatures 250C; carrier gas nitro-
gen (2 ml/min); detector dual FID; split ratio 1:30;
injection of 0.5 ll of a 10% hexane solution). The
identification of the components was performed,
for both the columns, by comparison of their
retention times with those of pure authentic
samples and by means of their linear retention
indices (l.r.i.) relative to the series of n-hydrocar-
bons. The relative proportions of the essential oil
constituents were percentages obtained by FID
peak-area normalization.
GC/EIMS analyses were performed with a
Varian CP-3800 gas-chromatograph equipped with
a DB-5 capillary column (30 m · 0.25 mm; coating
thickness 0.25 lm) and a Varian Saturn 2000 ion
trap mass detector. Analytical conditions: injector
and transfer line temperatures 220 and 240C
respectively; oven temperature programmed from
60C to 240C at 3C/min; carrier gas helium at 1
ml/min; injection of 0.2 ll (10% hexane solution);
split ratio 1:30. Identification of the constituents
was based on comparison of the retention times
with those of authentic samples, comparing their
linear retention indices relative to the series of n
hydrocarbons, and on computer matching against
commercial (NIST 98 and ADAMS) and home-
made library mass spectra built up from pure
substances and components of known oils and MS
literature data (Adams 1995, Davies 1990, Jennings
and Shibamoto 1980, Massada 1976, Stenhagen et
al. 1974, Swigar and Silverstein 1981). Moreover,
the molecular weights of all the identified sub-
stances were confirmed by GC/CIMS, using MeOH
as CI ionizing gas (same apparatus and analytical
conditions as above).
All the analyses were performed in triplicate.
The statistical analyses were performed using
SPSS 11.0.3 for Macintosh OS X running on a 1.66
GHz G4 machine.
All the concentrations were used as input data
(trace amounts were inserted as 0.001%) and no
treatment was performed before PCA processing.
Euclidean distances were used for MDS calcula-
tions. In the absence of information about shape of
the groupings and/or statistical distribution of
values, Euclidean distances combined with un-
weighted pair group average linking were used for
hierarchical cluster analysis (Dunlop et al. 1997).
Results and Discussion
The composition of the essential oils of the ten
Centaurea species is reported in Table 2.
Altogether, one-hundred and fifty com-
pounds have been identified. All the essential
oil samples are characterized by the presence of
sesquiterpenes, mainly hydrocarbon derivatives
and, in less amounts, oxygenated ones. Their
percentages accounted in almost all the samples
more than 60% of the whole oils. Among them,
the main ones were germacrene D, b-caryophyl-
lene, bicyclogermacrene, caryophyllene oxide,
spathulenol, a-copaene and, in lesser amounts,
but shared by all the species, a-cadinene,
a-muurolene, b-selinene and a-humulene.
Monoterpenes, when detected, were always
less than 1%. Among them, a- and b-pinene,
myrcene, a-phellandrene, p-cymene and limo-
nene were the most frequently identified ones.
Other common constituents were non-
terpene derivatives such as aliphatic aldehydes,
ketones, alcohols, esters and hydrocarbons.
Only the species C. iconiensis showed a very
different behavior: its essential oil resulted
composed by a small percentage of terpenes,
replaced by a high amount (84.3%) of the
unsaturated hydrocarbon 1-undecene, not de-
tected at all in all the other species, with the
exception of C. deflexa, but where it was
biosynthesized in considerable smaller
amounts (4.0%).
Taking into account the main constituents
of each essential oil, the ten species of Centau-
rea could be grouped as follows:
G. Flamini et al.: Centaurea classification using chemometric data 221

Table 2. Composition of the essential oils of ten Centaurea species from Turkey
Constituentsa l.r.i.b l.r.i.c 1d 2 3 4 5 6 7 8 9 10
e f
ethyl isovalerate 843 1154 – – – – – – – tr – –
ðEÞ-3-hexen-1-ol 852 1373 tr tr tr – – 1.4 0.3 tr – tr
ðEÞ-2-hexenal 854 1222 – – – 0.4 0.5 – – – tr –
ðEÞ-2-hexen-1-ol 862 1380 tr – – – tr – – – – –
2,6-dimethyl-1-heptene 866 844 – – – – – 3.8 – – – –
1-nonene 893 880 – – – – – – – 1.2 – –
2-heptanone 893 1175 – tr – – tr tr – – tr –
n-nonane 900 900 – – – 1.3 tr – – tr tr –
heptanal 906 1173 – tr tr 0.7 tr tr tr tr 0.3 –
anisole 920 1325 – – tr – – – – – – –
a-thujene 933 1020 – – tr – – – – – – –
a-pinene 941 1027 tr 0.7 1.6 1.7 tr tr 0.3 0.1 – tr
benzaldehyde 964 1497 – tr – tr tr tr – tr tr –
ðEÞ-2-heptenal 978 1240 – – – – tr – – – tr –
sabinene 978 1116 – tr 1.3 tr – – tr 0.1 – –
1-octen-3-ol 981 1391 – – – – tr – – – tr –
b-pinene 983 1108 – tr 1.1 tr – – tr 0.3 – –
6-methyl-5-hepten-2-one 987 1340 – – tr – tr tr – – – –
myrcene 992 1166 – – 2.0 0.7 0.3 – tr – – –
2-pentyl furan 993 – – 0.4 – 1.0 tr 0.8 0.4 – tr 0.6
1-decene 993 990 – – – – – – – 1.2 – –
mesitylene 996 994 – – – 0.4 – – – – – –
ðE; ZÞ-2,4-heptadienal 1001 1351 – – – – – – tr – – –
octanal 1003 1277 tr tr 0.3 0.4 tr tr tr tr 0.3 tr
a-phellandrene 1008 1167 – – tr – 0.3 – tr 0.5 – –
o-methyl anisole 1013 1349 – – tr – – – – – tr –
3-carene 1014 1144 tr – – – – – – – – –
ðE; EÞ-2,4-heptadienal 1017 1369 tr – tr 0.4 tr tr tr tr tr –
a-terpinene 1019 1180 – – tr – – – – – – –
p-cymene 1028 1243 – tr tr 6.1 tr – tr 0.2 – –
limonene 1033 1198 – tr 1.1 0.8 tr – tr 0.2 – –
b-phellandrene 1034 1205 – – tr – – – – – – –
1,8-cineole 1036 1209 – – tr – – – – – – –
ðZÞ-ocimene 1041 1240 – – tr – – – tr – – –
ðEÞ-ocimene 1051 1255 – – 2.0 – – – 0.6 tr – –
phenylacetaldehyde 1051 1618 tr tr – 0.4 – 1.5 – – tr –
c-terpinene 1063 1246 – tr 0.4 – – – – tr – –
ðEÞ-2-octenal 1064 1343 – – – – tr – – – tr –
acetophenone 1067 1465 – 0.7 – – – – – – – –
terpinolene 1089 1288 – – tr – – – – tr – –
1-undecene 1093 1084 – – – – – – 4.0 84.3 – –
p-cymenene 1093 – – – tr – – – – – – –
methyl benzoate 1094 1596 – – – – tr – – – – –
linalool 1101 1560 – – tr – – – – – – –
nonanal 1105 1382 0.2 1.7 2.9 0.4 0.4 tr 0.3 tr 0.7 0.6
heptanol acetate 1115 1377 – – – 7.1 – – – – – –
veratrole 1149 1753 – – – – – 0.8 tr – – –
cis-verbenol 1152 – – – tr – – – – – – –
ðE; ZÞ-2,6-nonadienal 1158 1573 – – tr – – – – – tr –
222 G. Flamini et al.: Centaurea classification using chemometric data

Table 2. (Continued)
Constituentsa l.r.i.b l.r.i.c 1 d
2 3 4 5 6 7 8 9 10
ðEÞ-2-nonenal 1165 1445 – – tr – tr – tr – tr –
pinocarvone 1168 – – – tr – – – – – – –
ethyl benzoate 1173 1645 – – – – – – – tr – –
4-terpineol 1181 1607 – 1.1 0.4 tr – – – tr – tr
p-cymen-8-ol 1187 1833 – – – – tr tr – 0.1 – –
1-dodecene 1193 1186 – – – – – – – tr – –
methyl salicylate 1194 1741 – – tr – – tr – tr – –
a-terpineol 1195 1684 – – – – – – – – – tr
n-dodecane 1200 1200 – – – – tr – – – tr –
dihydrocarveol 1200 – – – tr – – – – – – –
decanal 1207 1481 0.6 0.6 1.2 – 0.4 0.5 0.2 0.1 1.5 –
ðE; EÞ-2,4-nonadienal 1220 1597 – – tr – – – – – – –
methyl thymol 1236 1593 tr 2.1 – tr – tr tr – – –
hexyl 2-methylbutyrate 1236 – – – tr – – – – – – –
methyl carvacrol 1245 1606 – tr – – – – – – – –
hexyl 3-methylbutyrate 1245 – – – 0.3 – – – – – – –
ðEÞ-2-decenal 1264 1592 – – – – tr – – – – –
1-tridecene 1293 1289 – tr – – – – – 0.4 0.2 1.3
thymol 1293 2187 tr – – – – – – – – –
ðE; ZÞ-2,4-decadienal 1295 1708 – – tr – tr – – – – –
n-tridecane 1300 1300 – – – – – – – tr – –
undecanal 1305 1649 – – tr – tr – – – tr –
ðE; EÞ-2,4-decadienal 1319 1706 tr 0.6 0.3 – tr – tr tr – –
methyl decanoate 1327 1593 – – – – – – – – tr –
d-elemene 1339 1686 – – 0.4 tr – – – – – –
a-cubebene 1352 1461 – – tr – 0.3 – 0.2 – tr –
2,3,6-trimethylbenzadehyde 1365 – – 1.9 – 0.9 – – – – – –
cyclosativene 1371 – tr – tr – tr 0.5 0.4 – tr 3.0
a-ylangene 1373 1489 – – 1.6 – – – – – – –
a-copaene 1378 1477 0.8 0.7 0.8 1.1 0.9 1.5 3.4 – 0.8 2.7
b-bourbonene 1385 1517 – – – – – tr – – – –
b-cubebene 1390 1545 tr 0.6 tr 0.3 0.4 tr 0.7 – 0.5 –
b-elemene 1391 1593 – – – – – – tr – – –
1-tetradecene 1393 1386 – – – – – – – 0.1 tr –
ethyl decanoate 1396 1638 – – – – – – – tr – –
methyl 2,4-decadienoate 1398 – 0.8 – – – 3.2 – – – – –
n-tetradecane 1400 1400 – – – – – – – tr – –
methyl eugenol 1402 1757 – – tr – – – – – tr –
a-gurjunene 1408 1760 – – – – 0.4 – 0.2 – tr –
longifolene 1409 1577 tr – – – – – – – 0.3 –
thujaplicin 1411 – – – – – 0.8 – – – – –
p-2,5-dimetoxycymene 1417 – – – – – – 0.7 tr – – –
a-cedrene 1419 1568 tr – – 0.8 3.5 1.5 0.3 – tr –
b-caryophyllene 1420 1598 18.3 4.5 13.5 9.9 1.7 14.4 33.9 3.4 13.7 22.5
b-cedrene 1422 1638 tr 0.6 – 2.8 2.3 5.1 1.2 – tr –
b-gurjunene 1432 1590 – 1.0 – 2.2 0.4 – – – – 1.2
trans-a-bergamotene 1437 1782 tr – – – 0.6 0.4 – – – –
aromadendrene 1440 – – – – – – tr – – – –
G. Flamini et al.: Centaurea classification using chemometric data 223

Table 2. (Continued)
Constituentsa l.r.i.b l.r.i.c 1 d
2 3 4 5 6 7 8 9 10
ðEÞ-geranylacetone 1451 1842 – – 0.7 – – – 1.2 – – –
ðEÞ-b-farnesene 1456 1661 0.8 – 1.1 – 0.5 0.6 2.3 tr 0.7 –
a-humulene 1458 1665 1.7 1.9 1.0 1.4 0.7 1.4 2.7 0.1 1.8 3.0
b-santalene 1461 1648 – – – – – tr – – – –
cis-muurola-4(14),5-diene 1463 – – – – – – – – – tr –
b-acoradiene 1465 – – – – – – tr – – – –
ðEÞ-ethyl cinnamate 1468 2094 – – – – – – – 0.2 – –
c-muurolene 1478 1681 tr – 0.3 – tr 4.0 tr – tr –
germacrene D 1482 1712 22.7 45.1 40.2 43.0 40.2 21.7 21.2 – 43.1 36.9
b-selinene 1488 1715 1.7 1.0 1.6 0.8 0.6 3.7 0.5 tr 0.7 0.9
1-pentadecene 1492 1486 0.4 tr – 0.9 1.4 1.1 0.8 0.1 0.4 0.7
bicyclogermacrene 1496 1737 3.5 5.5 5.0 3.9 7.1 3.1 2.9 – 6.7 3.5
a-muurolene 1501 1711 0.3 – 0.3 – 0.2 0.5 0.4 – tr 1.6
ðE; EÞ-a-farnesene 1505 1727 0.4 – – – – – – – – –
b-bisabolene 1508 1708 1.3 1.0 – 3.2 – 1.1 0.9 – 1.2 1.7
tridecanal 1511 1822 0.5 – – – 0.9 – – – 0.8 –
c-cadinene 1514 1750 – – 0.5 – – tr 0.2 – tr –
d-cadinene 1522 1752 0.6 1.0 0.9 0.5 0.9 0.8 0.5 – 0.6 2.0
b-sesquiphellandrene 1526 1783 0.3 – – – – – – – 0.5 –
methyl dodecanoate 1527 1801 – – – – – – – tr – –
selina-3,7(11)-diene 1544 – – – – 0.5 – – – – tr –
trans-nerolidol 1565 2006 0.9 – – – 0.3 0.5 0.2 – 0.4 1.1
spathulenol 1578 2136 0.8 3.3 1.0 – 2.2 2.2 0.7 – 1.8 0.6
caryophyllene oxide 1584 1966 7.5 0.8 2.8 0.4 0.4 6.1 12.8 0.5 2.5 1.5
globulol 1586 2055 – 2.6 – – 1.1 – – – – 0.6
ethyl dodecanoate 1596 – – – – – – – – 0.1 – –
viridiflorol 1596 2104 0.3 – – – 0.5 – – – – –
guaiol 1598 – – – – – – – – 0.1 – –
n-hexadecane 1600 1600 – 0.5 – – – – – – – –
humulene epoxide II 1610 – 0.4 – – – – 0.4 0.6 – 0.2 –
1-epi-cubenol 1629 – – – 1.3 – 0.2 – – – – –
c-eudesmol 1633 2188 – – – – – – – – 0.5 –
T-cadinol 1644 2139 tr – 0.3 – 0.3 0.4 – – 0.3 –
T-muurolol 1646 2150 tr – 0.4 – 2.0 0.5 – – 0.3 –
a-muurolol 1649 – – – – – – – – – 0.2 –
b-eudesmol 1656 2249 11.8 – – – – – – – 4.7 –
a-eudesmol 1658 2233 tr 2.2 – 1.0 – 1.2 – 4.4 – 9.1
a-cadinol 1659 2188 – – 2.0 – 2.6 1.0 0.3 – – –
b-bisabolol 1673 2008 2.7 – – – 0.7 – – – 0.3 –
ðZÞ-a-santalool 1682 – – – – – – – – – 1.1 –
a-bisabolool 1687 2237 – 5.3 – – 1.9 – – – – –
1-heptadecene 1692 1689 – – – – 2.0 – – – 0.3 –
pentadecanal 1717 2060 0.9 – – – – – – – 0.7 –
ethyl tetradecanoate 1794 2044 – – – – – – – tr – –
octadecane 1800 1800 – – – – – – – tr – –
hexahydrofarnesylacetone 1843 2118 tr – – – – – – – – –
n-nonadecane 1900 1900 0.5 – – – – tr – 0.2 0.4 –
ethyl hexadecanoate 1994 2253 – – – – – – – 0.2 – –
224 G. Flamini et al.: Centaurea classification using chemometric data

Table 2. (Continued)
Constituentsa l.r.i.b l.r.i.c 1 d
2 3 4 5 6 7 8 9 10
n-eicosane 2000 2000 – – – – – – – 0.1 – –
n-heneicosane 2100 2100 4.7 – 0.3 – 0.3 tr – 0.5 1.2 –
n-tricosane 2300 2300 7.2 0.8 – – 0.9 tr 1.5 0.6 2.4 –
1-pentacosene 2492 2488 – – – – – – – – 0.4 –
n-pentacosane 2500 2500 1.8 – – – 2.5 – – – 1.9 –
a
Percentages obtained by FID peak-area normalization (mean of three analyses). Constituents are listed
according to their retention indices on a HP-5 column;
b
Linear retention indices (HP-5 column);
c
Linear retention indices (HP-WAX column);
d
1=C. aladaghensis, 2=C. antiochia var. prealta, 3=C. antitauri, 4=C. babylonica, 5=C. balsamita,
6=C. cheirolepidoides, 7=C. deflexa, 8=C. iconiensis, 9=C. lanigera, 10=C. ptosimopappoides;
e
not detected;
f
tr<0.1%.

C. babylonica, C. antitauri and C. lanigera
contained high concentrations of germacrene
D (43.0%, 40.2% and 43.1%, respectively) and
b-caryophyllene (9.9%, 13.5% and 13.7%,
respectively);
C. balsamita and C. antiochia had, similarly
to the previous three species, more than 40%
of germacrene D, but their content of b-
caryophyllene was significantly smaller (1.7%
and 4.5%, respectively). Other differences
could be referred to higher amounts of spath-
ulenol and lesser percentages of decanal;
C. cheirolepidoides and C. aladaghensis
were characterized by their lower content of
germacrene D (21.7% and 22.7%, respec-
tively), about half that of the above species,
but showed high contents of b-caryophyllene
(14.4% and 18.3%, respectively) and caryo-
phyllene oxide (6.1% and 7.5%);
C. deflexa contained a similar content of
germacrene D (21.2%) as observed in the
latter two species, but here b-caryophyllene
became the main constituent (33.9%), reach-
ing the highest percentage among all the ten
species. Also, the percentage of caryophyllene
oxide (12.8%) was the highest observed in this
study;
C. ptosimopappoides, with 36.9% of ger-
macrene D, was another species having high
amounts of this sesquiterpenic hydrocarbon,
but its percentage of b-caryophyllene (22.5%)
was quite high, about twice the content of the
other species (with the exception of C. deflexa);
C. iconiensis showed a very different chem-
ical behavior from all the other species, produc-
ing very high amounts of 1-undecene (84.3%),
followed by small percentages of b-caryophyl-
lene (3.4%) and caryophyllene oxide (0.5%).
The groupings resulted independent from
the ecological similarities (i.e. plants that live
in the same habitats or share similar morpho-
logical characteristics), so it seems that the
environment has no influence on the biosyn-
thesis of the volatiles of these plants.
To evaluate whether the identified essen-
tial oil constituents could be useful in reflect-
ing the taxonomic relationships among the
different species, the components of all the
essential oils were subjected to principal
component analysis (PCA), multidimensional
scaling (MDS) and hierarchical cluster anal-
ysis (HCA). In PCA the horizontal axis
explained about 78% of the total variance,
and the vertical axis a further 10.4%.
These statistical analyses (Figs. 1–3), where
all the compounds have been used for the
calculations, permitted to pointed out some
considerations. In fact, in the dendrogram and
in the biplots, C. iconiensis (sect. Centaurea)
was very far apart from all the other species.
The low loading on PC1 and the high loading
on PC2 for this species was due mainly to its
G. Flamini et al.: Centaurea classification using chemometric data 225

Fig. 1. a Principal Component Analysis (full plot) of ten Centaurea species from Turkey (al C. aladaghensis, ah
C. antiochia, at C. antitauri, by C. babylonica, bs C. balsamita, ch C. cheirolepidoides, de C. deflexa, ic
C. iconiensis, la C. lanigera, pt C. ptosimopappoides) b Principal Component Analysis (expanded plot) of
Centaurea species from Turkey. (al C. aladaghensis, ah C. antiochia, at C. antitauri, by C. babylonica, bs
C. balsamita, ch C. cheirolepidoides, la C. lanigera, pt C. ptosimopappoides)
226 G. Flamini et al.: Centaurea classification using chemometric data

Fig. 2. Multidimensional Scaling of ten Centaurea species from Turkey (al C. aladaghensis, ah C. antiochia, at
C. antitauri, by C. babylonica, bs C. balsamita, ch C. cheirolepidoides, de C. deflexa, ic C. iconiensis, la
C. lanigera, pt C. ptosimopappoides)

high content of 1-undecene (84.3%) (Fig. 1a). species (Fig. 1a). In MDS this situation was
The influence of the percentage of this com- confirmed by the very high positive score on
pound on PC2 loadings is also evidenced by C. dimension 1 for C. iconiensis and the high
deflexa, the only other species that produces 1- positive score on dimension 2 for C. deflexa
undecene (4.0%), that resulted much more (Fig. 2).
separated from the cluster of all the other

Fig. 3. Hierarchical Cluster Analysis of ten Centaurea species from Turkey (al C. aladaghensis, ah C. antiochia,
at C. antitauri, by C. babylonica, bs C. balsamita, ch C. cheirolepidoides, de C. deflexa, ic C. iconiensis, la
C. lanigera, pt C. ptosimopappoides)
G. Flamini et al.: Centaurea classification using chemometric data
This is in perfect agreement with the
recent proposal of transfer of C. iconiensis to
the genus Rhaponticoides by two different
botanists (Agabian 1997, Greuter 2003).
Another interesting finding emerging from
the statistical analyses is that C. lanigera,
from the Cyanus section, was deeply nested
in the dendrogram from the HCA analysis
with C. antitauri (sect. Pseudophaepappus)
and C. babylonica (sect. Microlophus), both
belonging to the Jacea group. These strict
correlations were also evidenced by the
biplots, where these species resulted very
close or almost overlapped because of the
highest positive loadings on PC1 and the
smallest negative ones on PC2, due to
the highest content of germacrene D
and the intermediate percentages of b-
caryophyllene (Figs. 1a and b). This is in
perfect agreement with the recently proposed
natural delimitation of the genus Centaurea
that should be limited to the species of the
Section Cyanus and of the Jacea group only
(Wagenitz and Hellwig 1996; Garcia-Jacas
et al. 2000, 2001).
Concluding, multivariate statistical analy-
ses applied to GC-MS data, seem to be very
useful to investigate and establish the natural
correlation within complex taxonomic groups.
The most useful technique seems to be HCA
because of its graphical representation (den-
drogram) that permits a much more clear
indication of the proximities among the differ-
ent species.
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Addresses of the authors: G. Flamini, (e-mail:
flamini@farm.unipi.it), M. Tebano, and P. L. Ci-
oni, Dipartimento di Chimica Bioorganica e Biof-
armacia, Via Bonanno 33, 56126 Pisa, Italy. Y.
Bagci, H. Dural, K. Ertugrul, and T. Uysal,
Department of Biology, Faculty of science and
Art, Selcuk University, Konya, Turkey. A. Savran,
Department of Biology, Faculty of Science and
Art, Nigde University, Nigde, Turkey.