Cent. Eur. J. Biol. • 8(8) • 2013 • 788-798
DOI: 10.2478/s11535-013-0174-5
Central European Journal of Biology
Anatomical characteristics and antioxidant
ability of Centaurea sadleriana reveals an
adaptation towards drought tolerance
Research Article
Jadranka Luković1, Djordje Malenčić2, Lana Zorić1,*, Miroslava Kodranov1, Dunja Karanović1,
Biljana Kiprovski2, Pal Boža1
1
Department of Biology and Ecology,
University of Novi Sad, Faculty of Sciences,
21000 Novi Sad, Serbia
2
Faculty of Agriculture,
University of Novi Sad,
21000 Novi Sad, Serbia
Received 07 November 2012; Accepted 27 February 2013
Abstract: The lamina, main vein and peduncle anatomical properties of Centaurea sadleriana Janka plants from two populations, were examined
using light and scanning electron microscopy. The indumentum was comprised of glandular and non-glandular trichomes of two
types. The leaves were amphistomatic, isolateral, with strongly developed palisade tissue. Secretory ducts were observed along the
phloem or sclerenchyma of large vascular bundles. Collenchyma alternated with chlorenchyma in the main vein and peduncle. Large
groups of strongly lignified sclerenchyma were present along the phloem of peduncle vascular bundles. These features, together
with thickened walls of epidermal cells and cuticle, numerous trichomes and thick-walled parenchyma in the perimedullar zone,
were perceived as a xeromorphic peduncle structural adaptation. Non-enzymatic antioxidant compounds of phenolic origin were
detected in small amounts and their respective content was higher in leaves compared to inflorescences. Compounds of phenolic
orgin showed positive correlation with total potenial of antioxidant activity indicated by the DPPH assay. Greater total quantity of
polyphenols and tannins was detected in leaves of plants from Zobnatica locality, while leaves of plants from Rimski Sanac were
characterized by higher content of total flavonoids and proantocyanidins. Phytochemical analysis showed that dominant secondary
biomolecules in inflorescences were phenolic pigments including anthocyanins and leucoanthocyanins, and free quinones in leaves.
Keywords: Leaf anatomy • Peduncle anatomy • Drought tolerance • Antioxidant activity • Asteraceae • Centaurea • Trichomes
© Versita Sp. z o.o.
1. Introduction
Genus Centaurea L. belongs to family Asteraceae and
comprises annual, biennial and perennial herbaceous
plants. In the flora of Europe and Serbia, 221 species
and 32 species have been recorded, respectively [1,2].
Ethnopharmacological studies reveal that many species
were well known for their use in traditional medicine and
for treatment of various diseases [3-5]. Owing to their
potential use in medicine, secondary metabolites isolated
from Centaurea species, as well as their biological
activities, were the subject of numerous investigations.
For this genus, the presence of sesquiterpene lactones,
flavonoids,
triterpenes,
acetylenes,
cyanogenic
glycosides, alkaloids and saponins is common. Some
788
samples also contained triterpene alcohols and lignans.
As Centaurea species were generally classified as
essential oil-poor plants, the investigations of these and
antioxidant properties are rather scarse. However, antiinflammatory, antimicrobial, antifungal and cytotoxic
activities had been found for extracts or natural products
of some of the Centaurea species [3,5-14].
All aerobic organisms possess antioxidant defence
mechanisms that provide balanced production
of reactive oxygen species (ROS). ROS include
superoxide radical (O2.-), hydroxyl radical (.OH), singlet
oxygen (1O2) and hydrogen peroxide (H2O2). They
are generated both in oxidative metabolism of normal
cells and during different stress-inducing situations.
Some of them include pathogen invasion, exposure to
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UV light and other forms of radiation, photooxidation,
air pollution, drought, herbicides, as well as following
certain injuries, hyperoxia, ozone, temperature
fluctuations and other stresses [15,16]. The main
cellular components susceptible to damage by free
radicals are polyunsaturated fatty acids in membranes,
proteins, carbohydrates, nucleic acids, and pigments,
such as chlorophyll or carotenoids [17]. Under normal
physiological conditions, the toxic effect of ROS is
suppressed by a strong antioxidant system consisting
of antioxidant enzymes (superoxide dismutase,
catalase, peroxidases, glutathione reductase, etc.) and
non-enzymatic components (proteins and peptides,
phenolics, carotenoids, etc.) [18].
A significant number of secondary biomolecules found
in natural products demonstrate distinct pharmacological,
aromatic, and antioxidant properties that make them
interesting for research and exploitation in pharmaceutical,
alimentary and cosmetic industry. Numerous studies
were carried out on many aromatic, spicy, medicinal and
non-medicinal plants that contain chemical compounds
exhibiting antioxidant properties, which resulted in a
development of natural antioxidant formulations for
nutritive, cosmetic, and other applications [19]. Since the
scientific information on antioxidant properties of various
plants, particularly those that are less widely used in food
preparation and medicine, is still lacking, the assessment
of such properties remains an interesting and useful task,
particularly for finding new sources for natural antioxidants,
functional foods and nutraceuticals [20].
Previous anatomical studies focusing on examining
structural characteristics of Centaurea species were
mostly performed by Turkish authors. Anatomical
descriptions were given for C. polyclada DC. [21],
C. glastifolia L. [22], C. calcitrapa L. ssp. cilicica (Boiss
& Bal.) Wagenitz and C. solstitialis L. ssp carneola
(Boiss.) Wagenitz [23], C. ptosimopappa Hayek
and C. ptosimopappoides Wagenitz [24]. Turkey,
with total of 114 endemic Centaurea taxa, has high
endemism ratio, which shows that this country is
one of the gene centers of this genus [21,24]. Gürdal
et al. [25] mentioned some anatomical properties of
leaf and stem in morphological description of C. kilaea
Boiss. and C. hermannii F. Hermann, endemic species
in European Turkey. More detailed anatomical analyses
were performed on C. rupestris L. and C. fritschii
Hayek by Rusak et al. [26], suggesting these two
species possess similar structure. The main anatomical
characteristics for species of Centaurea consist of the
presence of whip-like and glandular hairs, alternations
of chlorenchyma and collenchyma in stem and petiole,
presence of cortical and medullary vascular bundles
and occurrence of secretory ducts [27]. Among the
examined species, only C. fritschii had dorziventral
leaves, whereas other species had isolateral leaves,
with well developed palisade tissue. Secretory canals
were recorded in stem cortex, root endodermis and
secondary cortex, parenchymatous sheath of leaf
vascular bundles and above petiole vascular bundles in
C. rupestris and C. fritschii [26], as well as in root xylem
tissue and leaf mesophyll in C. polyclada [21].
C. sadleriana Janka is a perennial, herbaceous plant.
Although endemic to the Pannonian plane, in Europe it
is distributed in Austria, Hungary and Slovakia [2,28]. It
has been applied in Hungarian traditional medicine for
wound treatment in livestock, where its wound-healing
efficacy was shown [4]. Due to the fact that C. sadleriana
is biologically, phytochemically and pharmacologically
uninvestigated, the aim of this study was to investigate
micromorphological and anatomical characteristics,
antioxidant properties and phytochemical composition
of this species, collected from two different localities in
the north of Serbia. The aim was also to detect possible
differences in antioxidant ability between two native
populations of this species, as a result of adaptation to
different environmental conditions.
2. Experimental Procedures
Plant material was collected during the flowering period,
from two native populations (Zobnatica and Rimski
Sanac) in north Serbia. Both localities were steppe
habitats, had chernozem soil type and were exposed
to direct sunlight, except that Rimski Sanac locality had
lower precipitation during the growing season. Plants
were identified and voucher specimens deposited in the
Herbarium of the Department of Biology and Ecology,
Faculty of Sciences, University of Novi Sad (BUNS).
2.1 Anatomical analyses
For anatomical analyses, ten plants from each
population were fixed in 50% ethanol. The middle parts
of the leaves from the 6th node (lateral leaflet and the
main vein), as well as the middle parts of peduncles
carrying fully developed flowers, were separated. For
light microscopy observations, cross-sections were
prepared using a Leica CM 1850 cryostat, at a cutting
temperature of -20°C. Section thickness was 25 µm.
Leaf epidermal prints were obtained by covering the leaf
surfaces with liquid transparent varnish, and removing
the top layer by applying transparent adhesive tape.
Stomata were counted on five randomly selected
areas of the adaxial and abaxial surfaces, excluding
main veins, and calculated per mm2 of the leaf surface.
Measurements and observations were performed using
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Image Analyzing System Motic 2000. For scanning
electron microscopy (SEM) dry leaves were sputter
coated with gold for 180 seconds, 30 mA (BAL-TEC
SCD 005) and viewed with a JEOL JSM-6460LV electron
microscope at an acceleration voltage of 20 kV. As the
differences in the anatomical data were not significant
between the populations, data were presented as the
mean values of twenty plants.
2.2 Biochemical assays
Extracts of fresh and dry inflorescence and leaves from
10 plants per population were used for biochemical
analyses. All biochemical analyses were carried out
spectrophotometrically using a UV/VIS spectrophotometer
model 6105 (Jenway, UK). The production of superoxide
radical (O2.-) was measured by the inhibition of adrenaline
autooxidation [29] and expressed as nmol O2.- g-1 fresh
weight (fresh wt). Hydroxyl radical (.OH) production,
expressed as nmol .OH g-1 fresh wt, was determined by
the inhibition of deoxyribose degradation [30]. Superoxide
dismutase (SOD; EC 1.15.1.1) activity was measured by
monitoring the inhibition of nitroblue tetrazolioum (NBT)
reduction at 560 nm and expressed as U g-1 fresh wt
[31]. Lipid peroxidation (LP) was measured at 532 nm by
applying the thiobarbituric acid (TBA) test. The total amount
of TBARS (TBA-reactive substances) is given as nmol
malondialdehyde (MDA) equivalent of g-1 fresh wt [32].
Reduced glutathione (GSH) was determined according
to Sedlak & Lindsay [33] and expressed as µmol GSH g-1
fresh wt. Total polyphenol and tannin content was
determined following the Folin-Ciocalteu procedure
[34] and expressed as g catechin equivalent per g-1 dry
weight (d.wt). The amount of flavonoids was assessed
as described by Markham [35] and expressed as g
rutine equivalent per g-1 d.wt. Proanthocyanidins were
determined by a butanol-HCl assay and expressed as
mg leucoanthocyanidins g-1 d.w. [34]. Total potential
antioxidant activity of the investigated extracts was
assessed based on their scavenging activity of
1,1-diphenyl-2-picrylhydrazyl (DPPH) [36] and given as
% of neutralized radicals.
Phytochemical analyses were performed according to
Malenčić and Popović [37]. Previously prepared powder
and/or organic and aqueous extracts, were screened
for the presence of anthocyanins, leucoanthocyanins,
catechols, flavonoids, tannins, alkaloids, quinone
derivatives (free and conjugated), saponins, steroids,
and essential oils. The test results were qualitatively
expressed as negative (-) or positive (+).
2.3 Statistical analyses
The data were statistically processed using Statistica
for Windows (version 10.0, StatSoft, Inc. 2011). Relative
proportions of individual tissue types were calculated
and expressed as a ratio to the full lamina thickness or
full peduncle cross-section area. The general structure
of anatomical parameter variability was established by
performing Principal Component Analysis (PCA). Since
anatomical parameters had low variability and analysis
of variance showed that differences between the plants
from the two localities were negligible (data not shown),
the results of anatomical analyses for both localities
were presented together. However, due to difference in
antioxidant ability between populations from investigated
localities, these results were presented separately.
Values of the biochemical parameters were expressed
as means ± standard error of determinations made in
triplicates. Significance of differences in measured
parameters between the two populations was tested
using t-test (P<0.05).
3. Results
3.1 Leaf and peduncle anatomical characteristics
The lamina epidermis was composed of one layer of
large cells, with thickened outer walls, covered with
rugose cuticle. Adaxial and abaxial epiderms had similar
thicknesses, contributing 5.6 and 5.5% to the total lamina
thickness, respectively. The lamina was amphistomatic,
with stomata of the anomocytic type (Figure 1A).
Stomata were almost equally numerous on both lamina
surfaces, but smaller in size abaxially (Table 1). Nonglandular trichomes and glandular trichomes were
evidently more numerous on abaxial, and adaxial side,
respectively. Two types of non-glandular trichomes were
observed. Both were uniseriate, multicellular, with a very
long, thread-like terminal cell. The trichomes of the first
type had large, wide, thick-walled base cells, whereas
those of the second type had several smaller basal
cells, with thinner call walls (Figure 1C, D). Glandular
trichomes were sunken in the epidermis. They were
multicellular, composed of wide, flattened secretory
cells, above which a large subcuticular chamber was
observed (Figure 1B).
Lamina had an isolateral structure, with equally
well developed, two-layered palisade tissue on both
sides (Figure 2A). Palisade cells were cylindrical,
elongated, rich in chloroplasts and somewhat larger
adaxially. Spongy tissue cells of irregular shape were
arranged in one or two rows. Vascular bundles were
arranged in a line in the middle part of the mesophyll.
On average, 27 vascular bundles were observed in
leaflet cross-sections, occupying 2.5% of the entire
leaflet cross-section area. Amongst these bundles, only
two or three were significantly larger. All of the bundles
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A
B
C
D
Figure 1.
A - SEM micrograph of lamina adaxial epidermis (200x); B - Light micrograph of glandular trichome cross-section (600x); C - Light
micrograph of non-glandular trichome, type I (600x); D - Light micrograph of non-glandular trichome, type II (600x). bc – basal cell,
GL - glandular trichome, NGL I - non-glandular trichome of type I, NGL II – non-glandular trichome of type II, sc – secretory cells, sch –
subcuticular chamber, STO – stomata, tc – terminal cell.
were surrounded with parenchyma sheath cells that did
not contain chloroplasts. The sheath was especially
prominent around the large bundles, and connected with
collenchyma which appeared subepidermally in larger
veins. Along the phloem of large bundles secretory
ducts were observed.
The main vein had a heart shaped cross-section,
with two prominent ribs (Figure 2B). Epidermal cells
in this region had thickened walls and rugose cuticle.
Subepidermally collenchyma tissue was present on the
adaxial side, whereas, on the abaxial side, it alternated
with chlorenchyma tissue. Vascular bundles were
arranged in an arc. Three or four large vascular bundles
were observed, some with several small bundles between.
They were surrounded by well developed sclerenchyma
tissue. Along the sclerenchyma tissue by the phloem of
large bundles, secretory ducts were present. Percentages
of all main vein tissues, as well as the size of lamina cells,
all showed a high degree of variability.
Peduncle cross-section was polygonal in shape.
Epidermal cells had thickened walls and were
covered with thick cuticle and trichomes of the same
type as on the leaf. Prominent ribs contained groups
of collenchyma tissue (Figure 2C). Collenchyma
alternated with chlorenchyma, which was present
in larger groups between the ribs. Chlorenchyma
cells were usually arranged in 2-4 rows. One layer of
hypodermis was sometimes present between the ribs.
In cortex parenchyma, several small cortical vascular
bundles were present. In the central cylinder variable
numbers (8-17) of vascular bundles were arranged in a
circle. Large amounts of strongly lignified sclerenchyma
were present along the phloem of cortical and cylinder
bundles, sometimes almost completely surrounding
them. Parenchyma cells between the bundles in the
perimedullar zone also had very thickened cell walls.
The middle portion of the peduncle consisted of large
thin-walled parenchyma cells.
According to the results of the PCA analysis
anatomical parameters that defined the first axis, and
contributed to the total variation with 29.3%, were the
size of the main vein cross-section and palisade tissue
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Anatomy and antioxidant ability of Centaurea sadleriana
Anatomical character
mean ± S.E. (CV)
Lamina
Adaxial epidermis
Abaxial epidermis
Mesophyll
Cell cross-section area
Main vein
Cross section area (mm2)
2.2 ± 0.1 (23.8)
Cross section thickness (µm)
322 ± 8.1 (11.3)
Number of stomata/mm2
182 ± 4.9 (12.1)
Stomata length (µm)
24.8 ± 0.3 (6.1)
Stomata width (µm)
16.7 ± 0.3 (8.4)
% of epidermal thickness
5.6 ± 0.2 (16.9)
Number of stomata/mm2
197 ± 7.9 (18.0)
Stomata length (µm)
22.4 ± 0.4 (8.9)
Stomata width (µm)
13.6 ± 0.5 (15.0)
% of epidermal thickness
5.5 ± 0.2 (14.3)
% adaxial palisade tissue thickness
33.8 ± 0.9 (12.0)
% abaxial palisade tissue thickness
33.0 ± 0.9 (11.6)
% of spongy tissue thickness
23.6 ± 0.7 (13.8)
Number of vascular bundles
27.0 ± 0.8 (13.7)
% of vascular bundles
2.5 ± 0.2 (29.1)
Palisade tissue, adaxial (µm2)
766 ± 53.3 (31.1)
Palisade tissue, abaxial (µm2)
691 ± 41.2 (26.6)
Spongy tissue (µm2)
494 ± 35.8 (32.4)
Cross section area (mm2)
1.9 ± 0.2 (35.5)
% of parenchyma with epidermis
48.6 ± 1.8 (16.7)
% of collenchyma
5.7 ± 0.3 (21.7)
% of chlorenchyma
11.4 ± 1.2 (46.7)
% of vascular bundles
10.5 ± 0.8 (34.0)
% of sclerenchyma
9.1 ± 0.6 (28.4)
Cross section area (mm2)
2.2 ± 0.1 (24.9)
% of cortex parenchyma with epidermis
31.3 ± 1.0 (13.6)
Peduncle
Table 1.
% of collenchyma
7.2 ± 0.4 (22.1)
% of chlorenchyma
12.9 ± 0.4 (12.2)
% of vascular bundles
9.4 ± 0.4 (21.4)
% of sclerenchyma
11.8 ± 0.5 (19.2)
% of cylinder parenchyma
27.3 ± 0.8 (14.2)
Leaf and peduncle anatomical characteristics (mean, standard error and coefficient of variation %) (N=20).
cells, as well as percentages of spongy tissue, main
vein chlorenchyma and peduncle colenchyma. Other
anatomical parameters did not contribute significantly to
the total variability.
3.2 Antioxidant ability of C. sadleriana extracts
Determination of ROS production represents an
assessment of total antioxidant activity (enzymatic and
non-enzymatic) of fresh plant material extracts, i.e. the
ability of plant extracts to remove ROS efficiently. The
highest production of O2.- was registered in inflorescence
of the Rimski Sanac population and it was significantly
higher than that of the Zobnatica population. However,
as compared to the Rimski Sanac population, the
production of the most reactive oxygen intermedier, .OH,
was higher in inflorescences of Zobnatica population;
in particular, it was significantly higher in the leaves
(Figure 3).
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As for the results of SOD activity and lipid peroxidation
intensity in different organs of C. sadleriana from two
localities, there was no significant difference in SOD
activity between investigated populations. However, LP
intensity was significantly higher in both organs of the
Rimski Sanac population (Figure 4A, C).
Rimski Sanac
nmol ROS g-1 fr. w.
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
A
Zobnatica
*Ê
*Ê
I
L
I
Superoxide radical
Rimski Sanac
2000
1800
1600
1400
1200
1000
800
600
400
200
0
A
nmol MDA equivalents g-1 fr. w.
C
Figure 2.
Light micrographs of the cross-sections (100x): A –
lamina; B – main vein; C – peduncle. chl – chlorenchyma,
co – collenchyma, par – parenchyma, ph – phloem, pt –
palisade tissue, scl – sclerenchyma, sd – secretory duct,
vb – vascular bundle, xy – xylem.
180
160
140
120
100
80
60
40
20
0
C
Figure 4.
Zobnatica
Rimski Sanac
Zobnatica
80
70
% neutralized radicals
U g-1 fr. w.
B
Hydroxyl radical
ROS production in inflorescence (I) and leaves (L) of
investigated populations of C. sadleriana (the results
marked with an asterisk differ significantly at P>0.05).
I
L
60
*Ê
50
40
30
20
10
0
B
SOD
I
L
DPPH
60
*Ê
*Ê
I
L
LP
*Ê
50
μmol GSH g-1 fr. w.
Figure 3.
L
40
30
20
10
0
D
I
L
GSH
A - Superoxide dismutase activity (SOD), B - DPPHradical test (DPPH), C - lipid peroxidation intensity (LP),
D - reduced glutathione content (GSH) in inflorescence
(I) and leaves (L) of investigated populations of C.
sadleriana (the results marked with asterisk differ
significantly at P>0.05).
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Anatomy and antioxidant ability of Centaurea sadleriana
Regarding GSH and polyphenolics contents,
significant differences were registered only in leaves
of the C. sadleriana populations. The population from
Rimski Sanac had significantly higher GSH, total
flavonoid as well as proanthocyanidins content, whereas
our measurements revealed significantly higher content
of total polyphenols and taninns in plants from Zobnatica
(Figures 4D and 5).
The DPPH values for investigated extracts varied
over a wide range, i.e. between 11.4% and 66.3%
(Figure 4B). The inflorescence of plants from Zobnatica
was characterized by significantly higher percentage of
neutralized DPPH radicals.
Phytochemical screening of infusion and decoct of
inflorescences and leaves of investigated populations
of C. sadleriana showed dominant presence of
anthocyanins, leucoanthocyanins and free quinone
derivatives (Table 2).
the type and density of epidermal indumentum of leaf and
stem were of particular taxonomic value. We recorded
two types of non-glandular trichomes on C. sadleriana,
including those with thick-walled and thin-walled basal
cells, which were more numerous on the abaxial
epidermis. Rusak et al. [26] found the same two types
on the leaves of C. rupestris and C. fritschii. According
to Metcalf and Chalk [27], non-glandular trichomes with
uniseriate pedestal and long, whip-like terminal cell are
very common in this genus. Thin-walled multicellular
covering hairs with 5-9 basal cells in C. kilaea or 7-14
cells in C. hermannii were recorded by Gürdal et al. [25].
Rimski Sanac
Zobnatica
(I)
(L)
(I)
(L)
Anthocyanins (i)
+++
-
+++
-
Leucoanthocyanins (i)
+++
-
+++
-
Catechols (i)
-
+
-
+
4. Discussion
Flavonoids (i)
++
++
++
+
Tannins (i)
+
++
+
++
Several species from the genus Centaurea are interesting
from medicinal and pharmacological perspective, and
thus recently the subject of intensive research [3-14].
Since structural and phytochemical characteristics of
C. sadleriana were previously unknown, our work
highlights this information. Our findings report that the
anatomy of C. sadleriana lamina resembled that of other
Centaurea species [21-23,25,26] however we provide
a detailed descriptions of the main vein and peduncle
anatomy.
Rahiminejad et al. [38] found that, among
micromorphological characters of Centaurea species,
Alkaloids-Mayer’s reagent (i)
-
++
-
+
Alkaloids-Dragendorff’s reagent (i)
Quinone derivatives-free (i)
90
-
+
+
+++
-
-
-
-
Saponins (d)
-
+
-
+
Steroids (d)
-
-
-
-
Essential oils (i)
-
-
-
-
Phytochemical screening of infusion (i) and decoct (d) of
inflorescences (I) and leaves (L) of investigated populations
of C. sadleriana ((-) absence, (+) low concentration, (++)
moderate concentration, (+++) high concentration).
Zobnatica
*Ê
80
70
mg g-1 d. w.
++
+++
Quinone derivatives-conjugated (i)
Table 2.
Rimski Sanac
++
*Ê
60
50
40
30
*Ê
20
10
0
*Ê
I
L
TP
Figure 5.
I
L
TT
I
L
TF
I
L
PRO
Contents of phenolics compounds (TF - total polyphenols, TT - total tannin, TF - total flavonoids, PRO - proanthocyanidins) in inflorescence
(I) and leaves (L) of investigated populations of C. sadleriana (the results marked with asterisk differ significantly at P>0.05).
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Similarly, Altundag and Gürdal [22] found unicellular
and multicellular hairs on C. glastifolia. According to
Uysal et al. [21] C. polyclada had one or two to three
celled hairs on leaves, which were not confirmed in our
investigations for C. sadleriana. Although the presence
of glandular trichomes was mentioned by several
authors, their structure was not described previously
for this genus. Rusak et al. [26] provided micrographs
of elongated biseriate hair from florets of C. fritschii
and glandular hair from C. rupestris stem. As a part
of this work, on examined species we recorded one
type of glandular trichomes, which were sunken in the
epidermis. They were more numerous on the adaxial
epidermis, and composed of several flattened secretory
cells, with large subcuticular chamber above them.
As in most of the Centaurea species, the leaves
of C. sadleriana were isolateral, with two layers of
palisade cells on both sides, which is characteristic for
plants that inhabit dry and well insolated habitats [39].
Anatomical parameters that were previously singled out
as characteristic for Centaurea species by Metcalfe and
Chalk [27] and Rusak et al. [26] were also observed in
C. sadleriana. Those were alternations of chlorenchyma
and collenchyma in the main vein, petiole and peduncle,
presence of cortical and medullary vascular bundles
in stems and occurrence of secretory ducts. Secretory
canals were recorded along the phloem or phloem
sclerenchyma of leaf vascular bundles. Especially
prominent were large groups of lignified sclerenchyma,
which almost completely surrounded the peduncle
vascular bundles. Presence of thickened, rugose cuticle,
thickened epidermal cell walls, numerous trichomes,
significant amounts of strongly lignified sclerenchyma
and thick-walled parenchyma in perimedullar zone could
thus be recognized as xeromorphic peduncle structural
adaptations.
Intensive metabolic processes at the time of plant
collecting (July, a period of full blossoming), as well as
unfavourable environmental factors, such as drought,
UV-radiation and high temperatures, could provoke
intense ROS production [40]. According to our results,
plant extracts from Zobnatica locality were more efficient
at removing O2.-, whereas these from Rimski Sanac
efficiently removed .OH.
SOD activity was established in both specimens of
C. sadleriana. It has been also determined in several
other cultivated and wild growing species, such as in
Allium sativum (803.37 U g-1 fr.w.), Achillea millefolium
(399.09 U g-1 fr.w.), Salvia reflexa (506.9 U g-1 fr.w.)
[41], S. glutinosa (620.54 U g-1 fr.w.) and S. nemorosa
(125.09 U g-1 fr.w.) [42], Ruscus hipoglossum (263.7423.6 U g-1 fr.w.), Lillium martagon (32.42 U g-1 fr.w.),
etc. In comparison to these findings, SOD activity in C.
sadleriana was significantly higher, especially in leaves.
Although elevated SOD activity was expected, due to
intensive photosynthetic activity in the full blossoming
stage, when O2.- is being generated in excess, this trait
may also be genetically determined.
Lipid peroxidation (LP) is a reliable indicator of
oxidative stress, marked as the main cell damage
mechanism in many biological systems of plant and
animal origin. ROS generated in a cell may react with
unsaturated fatty acids, causing peroxidation of lipid
membranes in plasmalemma and/or cell organelles,
leading to cell leakage, rapid desiccation and cell death
[18]. Being an end-product of LP, malondialdehyde
(MDA), together with other thiobarbituric acid-reactive
substances (TBARS), represent biomarkers of oxidative
stress. Our results show that LP was more pronounced
in the inflorescence in both populations, especially
the one from Rimski Sanac locality. This observation
is in line with the results obtained for O2.- production.
Increased production of O2.- in inflorescences of plants
collected from this locality may have led to generation
of other toxic ROS, which resulted in cell membrane
destruction and most intensive LP.
In addition to enzymes, plant`s antioxidant responses
also involve non-enzymatic protective systems, such
as reduced glutathione, carotenoids, flavonoids,
ascorbate and tocopherols. These “small molecules”
may compensate, in full or partially, for lower enzyme
activity. In C. sadleriana populations high content of
reduced glutathione (GSH) has been established. GSH
is engaged in the process of ROS elimination in cells
of aerobic organisms, whereas plant phenolics play an
important role in LP stabilization and are closely linked
to antioxidant activity [43].
Joyce et al. [44] conducted research on C. jacea,
confirming high content of total polyphenols (TP)
and high antioxidant capacity. In our specimens
of C. sadleriana, the TP content, as well as that of
particular classes of phenolics, was low. It has also
been established that leaves contained more phenolics
compared to inflorescences, especially TP and TT in
the Zobnatica population. The population from Rimski
Sanac showed significantly higher content of flavonoids
and proanthocyanidins.
Inflorescences from Zobnatica population showed
significantly higher percentage of neutralized radicals
compared to representatives of the other population.
Leaf extracts showed higher activities then inflorescence
extracts, and could be classified as moderately active.
These results are in accordance with those obtained
for non-enzymatic antioxidants contents, especially in
leaves, which could have led to the higher DPPH-radical
scavenging activity. Higher levels of DPPH activity
795
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Anatomy and antioxidant ability of Centaurea sadleriana
have been correlated with tolerance to different stress
conditions [45], but they also point out to the presence
of biologically active biomolecules with pronounced
antioxidant activity [15].
The presence of flavonoids was detected in
both populations, with the dominant presence of
anthocyanins and leucoanthocyanins in inflorescences,
and free quinones in leaves. Saponins and catechols
are present in small amounts in leaves, and tannins
and alkaloids in inflorescences. Although not fully
investigated, presence of saponins in genus Centaurea
was also established in C. gloriosa var. multiflora Radić
[46] and C. squarosa [47]. The presence of alkaloids
was determined in many representatives of this genus,
such as in C. macrocephala Puschk., C. arenaria M.
Bieb., C. transcaucasica D.Sosn., C. micranta Dufour.,
C. stereophylla Bess., C. repens L. and C. solstitialis
L. [48]. However, the presence of conjugated quinone
derivatives, steroids and essential oils has not been
established.
The results obtained for the antioxidant
characteristics of C. sadleriana from the southern parts
of the Pannonian plain showed that both populations
had moderate overall antioxidant ability. Nonenzymatic antioxidant compounds of phenolic origin
were detected in smaller amounts and their content
was higher in leaves compared to inflorescences,
which was positively correlated with the results of the
DPPH assay.
Phytochemical analysis showed that dominant
secondary biomolecules in inflorescences were phenolic
pigments - anthocyanins and leucoanthocyanins, and
free quinones in leaves. Xeromorphic characteristics
of leaves and peduncles, as well as higher activity of
antioxidant protective mechanisms in leaves of plants
from the Rimski Sanac population, point to unfavourable
environmental conditions plants were exposed to. Thus,
further monitoring of the endemic and endangered
species that grow wild in the southern parts of the
Pannonian plain is recommended.
Acknowledgements
The authors would like to thank Mr. Milos Bokorov from
University Center for Electron Microscopy, Novi Sad,
for his technical assistance and SEM microscopy.
This work was financially supported by the Ministry
of Education and Science, Republic of Serbia, Grant
No. 173002.
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