Antioxidative responses to seasonal changes and chemiluminescence assay of Astragalus onobrychis leaves extract

Abstract

The aim of this study was to research the seasonal changes of antioxidant enzyme activity and total antioxidant capacity in leaves of Astragalus onobrychis L. subsp. chlorocarpus (Griseb.) S. Kozuharov et D.K. Pavlova. Leaves of A. onobrychis were collected during the different stages of growth and analyzed for antioxidant enzyme activity: superoxide dismutase, catalase, guaiacol peroxidase, glutathione peroxidase. Quantities of malonyldialdehyde, superoxide radicals, and hydroxyl radicals were measured as well as the content of soluble proteins. Furthermore, total antioxidant capacity was determined by the inhibition of chemiluminescence activity of blood phagocytes by leaf extracts. Stages of vegetation significantly affected the accumulation of superoxide radicals, but there were no significant differences in hydroxyl radical quantity and lipid peroxidation levels during vegetation. Soluble proteins vary greatly between different stages of growth. Seasonal changes were found to have an effect on enzymatic activities. During the spring season, guaiacol peroxidase showed the highest levels. Catalase and glutathione peroxidase increased their activities in summer, while, during the autumn season, superoxide dismutase showed maximum activity. On the basis of chemiluminescence assay, it can be concluded that leaf extract of A. onobrychis possesses a significant antioxidant capacity thus protecting plants during environmental stress.

Full text

1. Introduction
Environmental stresses, as well as developmental
processes, induce the production of reactive oxygen
species (ROS) in a plant. ROS have long been proposed
as signal molecules that regulate various processes
such as growth, development, and responses to
biotic and abiotic stress factors [1]. However, at high
concentrations, ROS can be toxic by destroying normal
metabolism through oxidative damage to lipids, proteins
and nucleic acids [2]. Since they are involved in ROS
metabolism, antioxidant enzymes may play an important
part in the plants’ strategies of tolerance allowing survival
in their habitats [3]. The most important components of
the antioxidant system include several enzymes: those
that directly eliminate ROS (e.g. superoxide dismutase,
catalase and peroxidase) and enzymes that eliminate
internal lipid peroxidation products (e.g. glutathione
Central European Journal of Chemistry
* E-mail: dragoljubm@gmail.com
1Department of Pharmacy, Faculty of Medicine, University of Niš,
18000 Niš, Serbia
2Department of Biology, Faculty of Medicine, University of Niš,
18000 Niš, Serbia
3Center of Chemistry - IChTM, University of Belgrade,
Njegoševa 12, 11000 Belgrade, Serbia
4Laboratory for Geochemistry, Cosmochemistry and Astrochemistry,
University of Niš, 18000 Niš, Serbia
Dragoljub L. Miladinović1*, Budimir S. Ilić1,
Stevo J. Najman2, Olga G. Cvetković3, Aleksandra M. Šajnović3,
Marija D. Miladinović1, Nikola D. Nikolić4
Antioxidative responses to seasonal
changes and chemiluminescence assay
of Astragalus onobrychis leaves extract
Invited Paper
Abstract:
© Versita Sp. z o.o.
Received 9 July 2012; Accepted 21 October 2012
Keywords: Astragalus onobrychis • Antioxidant enzymes • Reactive oxygen species • Antioxidant capacity • Chemiluminescence
The aim of this study was to research the seasonal changes of antioxidant enzyme activity and total antioxidant capacity in leaves of
Astragalus onobrychis L. subsp. chlorocarpus (Griseb.) S. Kozuharov et D.K. Pavlova. Leaves of A. onobrychis were collected during
the different stages of growth and analyzed for antioxidant enzyme activity: superoxide dismutase, catalase, guaiacol peroxidase,
glutathione peroxidase. Quantities of malonyldialdehyde, superoxide radicals, and hydroxyl radicals were measured as well as the
content of soluble proteins. Furthermore, total antioxidant capacity was determined by the inhibition of chemiluminescence activity
of blood phagocytes by leaf extracts. Stages of vegetation signicantly affected the accumulation of superoxide radicals, but there
were no signicant differences in hydroxyl radical quantity and lipid peroxidation levels during vegetation. Soluble proteins vary greatly
between different stages of growth. Seasonal changes were found to have an effect on enzymatic activities. During the spring season,
guaiacol peroxidase showed the highest levels. Catalase and glutathione peroxidase increased their activities in summer, while, during
the autumn season, superoxide dismutase showed maximum activity. On the basis of chemiluminescence assay, it can be concluded
that leaf extract of A. onobrychis possesses a signicant antioxidant capacity thus protecting plants during environmental stress.
Cent. Eur. J. Chem. • 11(2) • 2013 • 123-132
DOI: 10.2478/s11532-012-0163-6
123

Antioxidative responses to seasonal changes and chemiluminescence
assay of
Astragalus onobrychis
leaves extract
peroxidases) [4]. To add more evidence to the ecological
studies already carried out on wild plant adaptation, it is
of importance to understand the molecular, biochemical
and physiological means which protect plants from
environmental adversities.
Formation of activated oxygen compounds occurs
during respiratory burst of phagocytic cells with the
generation of photons that produce a very weak light
signal, which may be amplied and measured as
chemiluminescence (CL). The generation of CL has
been reported to involve superoxide, singlet oxygen,
hydrogen peroxide and hydroxyl radicals. Therefore, CL
method is commonly used for measuring antioxidative
properties of plant extracts.
Astragalus L. (Fabaceae) is generally considered
the largest genus of vascular plants with an estimated
number of 2500−3000 species [5]. Many species of
Astragalus are used to restore overgrazed range, control
erosion, and provide useful sources for producing
important drugs [6]. The ora of Serbia includes
seventeen species of the Astragalus genus [7].
Our previous study dealt with the mineral and
non-enzymatic antioxidant composition of Astragalus
onobrychis L. subsp. chlorocarpus (Griseb.) S.
Kozuharov et D.K. Pavlova [8]. This research was
designed to study the changes of enzymatic antioxidants
in leaves of A. onobrychis from Serbia during the active
vegetative period. We investigated the activities of
antioxidant enzymes: superoxide dismutase (SOD),
catalase (CAT), guaiacol peroxidase (POD), glutathione
peroxidase (GPOX); quantities of malonyldialdehyde
(MDA), superoxide (O2•-), hydroxyl (• OH) radicals and
the content of soluble proteins were also measured. The
paper also describes the total antioxidant capacity (TAC)
determined by the inhibition of CL activity of mice blood
phagocytes by leaf extracts. To the authors’ knowledge,
the changes of the enzymatic antioxidative system
related to the stage of vegetation and TAC determination
by in vitro assay in leaves of A. onobrychis have not
been previously examined.
2. Experimental procedure
2.1. Chemicals
All chemicals and reagents were of analytical reagent
grade and were purchased from the Sigma–Aldrich
Chemical Company.
2.2. Plant material
Astragalus onobrychis L. subsp. chlorocarpus (Griseb.) S.
Kozuharov et D.K. Pavlova grows among communities of
sub-Mediterranean grasslands Astragalo-Potentilletalia
and Scabioso-Trifolion dalmatici in phytocoenoses
which is described as the Astragalo-Calaminthetum
hungaricae [7].The leaves were collected from healthy
specimen in their natural habitat at the Seličevica
mountain in 2011. The plant material was collected in
three stages of growth (SG) as follows:
- 1st SG – the initial vegetation stage, spring (25 April)
- 2nd SG – the blooming stage, summer (7 July)
- 3rd SG – the seed forming stage, autumn (2 October)
Astragalus onobrychis leaves have been picked
between 15:00 and 16:00 h when sun insolation was
highest. Fresh leaves were immediately frozen in liquid
nitrogen and transported to the laboratory where they
have been stored at -70°C until preparation of extracts
began.
Botanical identication was made by Dr. N.
Ranđelović at the Botany Department, Faculty of
Science, University of Niš, Serbia, where a voucher
specimen is deposited.
Characteristics of soil were investigated by standard
analytical methods and techniques [9]. Observed
meteorological parameters were obtained by the State
Hydrometeorological Service, from the meteorological
station located in Niš (Table 1).
2.3. Antioxidants analysis
2.3.1. Extraction of enzymes
One gram of fresh leaves were ground with quartz sand
in a cold mortar. The ground material was suspended
in 5 mL K2HPO4 (0.1 mol L-1) at pH 7.2. After 10 min
centrifugation at 4°C and 15000 × g, the aliquots of
the supernatant were used for antioxidant activity
determination.
2.3.2. Determination of quantities MDA, O2•- and • OH
Lipid peroxidation (LP) was determined by the
thiobarbituric acid (TBA) method [10]. A 0.5 mL aliquot of
supernatant was mixed with 2 mL of 20% trichloroacetic
acid (TCA) containing 0.5% TBA. The mixture was
heated at 95oC for 30 min, quickly cooled, and then
centrifuged at 15000 × g for 10 min. The absorbance
of the supernatant at 532 nm was read and the value
for non-specic absorption at 600 nm was subtracted.
Values were given as equivalent amounts of MDA. The
calibration curve was prepared with malonyldialdehyde
bis-diacetal. O2•- was determined by adrenaline
autooxidation [11], while •OH was determined by the
inhibition of deoxyribose degradation [12].
2.3.3. Determination of antioxidant enzyme activity and
protein content
All the antioxidant enzyme activities were determined
spectrophotometrically at 25°C using phosphate buffer
124

D. L. Miladinović et al.
(pH 7.2) plant extracts. Enzymatic specic activity is
expressed as μmol of the substrate transformed/min/mg
protein except for superoxide dismutase activity.
Superoxide dismutase activity (SOD, EC 1.15.1.1)
was determined by the method of Misra and Fridovich
[11] based on the inhibition of transformation of
adrenaline to adrenochrome at pH 10.2. One unit SOD
can be regarded as that amount of enzyme which
causes a 50% inhibition in the extinction change in
1 min compared with the control. Measurements were
made at 480 nm. Catalase activity (CAT, EC 1.11.1.6)
was determined at 240 nm. The decomposition of
H2O2 was followed by a decrease in absorbance [13].
Guaiacol peroxidase activity (POD, EC 1.11.1.7.)
was determined using guaiacol as the substrate
at 436 nm [14]. Glutathione peroxidase activity
(GPOX, EC 1.11.1.9.) was determined using cumene
hydroperoxide and reduced glutathione (GSH) as
substrates at 412 nm [15]. The soluble protein content
was determined by the method of Bradford with bovine
serum albumin as standard [16].
2.4. CL assay
The CL assay was performed to measure the TAC of
plant origin. CL intensity of mice blood leukocytes served
as a degree of oxidant activity [17]. Luminol (5-amino-
2,3-dihydrophthalazine-1,4-dione) solution was used as
a chemiluminegenic probe. Butyl-hydroxytoluene (BHT),
a common antioxidant, was used as a standard.
2.4.1. Blood samples
Polymorphonuclear leukocytes (PMNs) were obtained
from male BALB/c mice as previously reported [18].
Venous blood was centrifuged initially at 170g to
remove the platelet-rich plasma and then at 1000g to
eliminate platelet-poor plasma. The buffy coat of white
cells were diluted with phosphate buffered saline (PBS)
pH 7.2. Dextran was added and the mixture was left for
45 min at room temperature (26°C) for sedimentation.
The supernatant was centrifuged by Ficoll-gradient
separation and then washed twice with distilled water to
remove red blood cells. A pellet of PMNs was collected
from the tube base. The cells were suspended in Hanks
Balance Salt Solution (HBSS) pH 7.4 with Ca2+ and
Mg2+ (HBSS++). Cell suspensions were counted using
a hemocytometer and light microscope, and they were
then diluted with HBSS to obtain a nal cell suspension
of 1×106 mL-1.
The sample was then taken to calculate the leukocyte
formula and the absolute number of PMNs. Blood
sample without testing extracts served as a control. The
study was performed according to the guidelines of the
Ethical Committee of the Faculty of Medicine, University
of Niš, Serbia, which is in accordance with international
regulations.
2.4.2. Cell viability
Cell viability was determined by the standard trypan
blue exclusion method. The PMNs (1×106 mL-1) were
incubated with 3.5 or 7.0 mg mL-1 of plant extracts each
in triplicate at room temperature for 2 h. The blue dye
uptake was an indication of cell death. The percentage
viability was calculated from the total cell counts [19].
2.4.3. CL measurement
CL measurements were performed using a liquid
scintillation counter (Beckman LS 3200) in the
coincidence off-mode. The measurement was carried
out in glass vials, which previously read “background”
CL, to remove vials with high cpm. The samples were
measured in duplicate, every 5-10 minutes (repeated
circle), with a required reading of the sample without
stimulators (unstimulated CL) for 2 hours.
150 mL of blood, phosphate buffered saline pH
7.2, luminol and stimulator of phagocytes for a total
volume of 1500 mL were placed in measured vials.
Luminol, 0.1 mol L-1 in dimethylsulfoxide (DMSO), was
adjusted according to the number of PMNs in the blood
sample to be measured. 15 mL of Zymosan was used
as the stimulator. For unstimulated chemiluminescence,
PBS was added to a volume, instead of zymosan,
up to 1500 mL. The measurement began with the
addition of luminol solution to the diluted blood with a
calculated amount of PBS. CL response of phagocytes
was induced by placing the zymosan solution into the
measuring sample.
In order to estimate antioxidant compounds of A.
onobrychis, two ways of extraction preparation were
used. Crude leaf extract was prepared at the same time
for antioxidant activity determination. Boiled leaf extract
was retrieved after cooling of the crude leaves (95°C,
30 minutes). Leaves collected in the blooming stage of
vegetation were used.
Table 1. Observed meteorological parameters over the natural
habitat of A. onobrychis.
Season
Air
temperature
(°C)
Average
sunlight
hours/day
Rainfall
(mm)
Spring 11.94±1.78 5.72±0.64 1.24±0.16
Summer 22.98±2.14 9.10±0.97 1.25±0.13
Autumn 12.07±1.06 5.98±0.49 0.84±0.08
125

Antioxidative responses to seasonal changes and chemiluminescence
assay of
Astragalus onobrychis
leaves extract
2.5. Data analysis
The experimental results of antioxidant analyses were
expressed as a mean ± standard deviation of three
replicates. The comparison of biochemical parameters
was analyzed by ANOVA and Tukey test using the
statistical program SPSS v19.0 (SPSS Inc., Chicago,
IL, USA). The statistical signicance for all tests were
set at the P≤0.05 condence level. Bivariate correlations
of Pearson were used to study the interaction of studied
biochemical parameters.
3. Results and discussion
3.1. Environmental conditions at A. onobrychis
habitat
Soil parameters pH (7.78), redox potential (122.47 mV),
and humus (1.41%) were consistent with the results
of our previous studies and did not differ signicantly
from the investigated soils of south-eastern Serbia [20].
Temperature interval for the year was rather typical,
with average temperature in summer two-fold higher
than that in spring and autumn. Sunlight hours followed
seasonal dynamics, with a maximum in summer (9.10
average sunlight hours/day). As for average season
rainfall, values for spring (1.24 mm), summer (1.25 mm)
and autumn (0.84 mm) showed dryer seasons than
normal (Table 1).
3.2. O2•-, • OH, and MDA quantities
The highest values of LP, measured as MDA equivalents
and O2•- (Table 2), were recorded in the seed forming
stage, while the highest • OH accumulation was observed
in the blooming stage. Stages of vegetation affected the
accumulation of O2•- signicantly (F = 372.20), but there
are no signicant differences in • OH quantity (F = 0.25)
and LP levels (F = 4.46) during vegetation. An increase
of MDA quantity during vegetation was correlated with
a signicant increase of O2•- (r = 0.82; Fig. 1A) and
a change of •OH (r = 0.70; Fig. 1B) leaf quantities,
respectively.
3.3. Protein content and antioxidant enzyme
activities
Soluble proteins vary greatly between different stages
of growth, and the values range from 5.24 mg g-1 and
10.35 mg g-1 (Table 2). The highest level was recorded
in the blooming vegetation stage. Signicant differences
in protein content during vegetation (F = 35.52) can be
related to O2•- quantities in leaves (r = - 0.75; Fig. 2).
Vegetation stage had a signicant effect on
antioxidant enzymes activity in leaves of A. onobrychis
(SOD, F = 15.79; CAT, F = 7.83; POD, F = 5.25; GPOX,
F = 18.14; Table 2). The activity of SOD was higher in
the seed forming stage (46.73 U mg-1 protein), than
in the other two vegetation stages. The highest CAT
activity was recorded in the blooming stage of vegetation
(7.93 U mg-1 protein), similar to GPOX activity
(0.45 U mg-1 protein). The highest value of POD activity
was noted in the initial vegetation stage (8.13 U mg-1
protein).
CAT and POD activities were related to protein
concentrations (r = 0.94, r = 0.76; Fig. 3A and 3B).
In contrast, the changes in CAT and POD activities,
can be associated with the increase of O2•- quantity
(r = - 0.74, r = - 0.74; Fig. 3C and 3D). Negative correlation
coefcients indicate an active antioxidant role of CAT
and POD in protection against O2•- radicals.
3.4. Total antioxidant capacity
The cell viability test carried out to evaluate the cytotoxicity
of plant extracts on mice blood phagocytes at 3.5 and
7.0 mg mL-1 indicated that cells were viable (>90%) after
2 h incubation. It is known that phagocytosis and other
Table 2. Quantities of O2
•-, •OH, MDA, protein contents and activities of antioxidant enzymes in leaves of A. onobrychis.
Biochemical parameters Stages of growth F
1st 2nd 3rd
O2
•- (nmol mg-1 protein) 123.78 ± 7.78 163.72 ± 8.19 312.48 ± 10.56 372.20*
•OH (nmol mg-1 protein) 1.76 ± 0.27 1.92 ± 0.31 1.87 ± 0.26 0.25
MDA (nmol mg-1 protein) 9.93 ± 0.89 11.37 ± 1.94 14.02 ± 2.03 4.46
Protein (mg g-1)8.21 ± 0.64 10.35 ± 0.90 5.24 ± 0.67 35.52*
SOD (U mg-1 protein) 42.72 ± 4.87 27.18 ± 3.28 46.73 ± 5.13 15.79*
CAT (U mg-1 protein) 7.52 ± 0.68 7.93 ± 0.72 5.88 ± 0.61 7.83*
POD (U mg-1 protein) 8.13 ± 0.42 7.86 ± 0.48 7.08 ± 0.32 5.25*
GPOX (U mg-1 protein) 0.32 ± 0.02 0.45 ± 0.03 0.41 ± 0.03 18.14*
F - ratio between groups variance and the variance within groups; * - results of the variance analysis, where the seasonal changes effect is signicant.
126

D. L. Miladinović et al.
states of phagocyte stimulation are the source of very
active oxidants [21]. The parameters of the inhibitory CL
activity of blood phagocytes by A. onobrychis extracts
are given in Table 3 and Figs. 4 and 5. Based on the
measurement of the area over the time course of the CL
curves, we estimated the CL inhibition of plant extracts.
In relation to the CL inhibition of the same concentration
of BHT, an index of inhibition was calculated. Common
antioxidant BHT showed higher CL inhibition, compared
with plant extracts. According to the peak values of the
proportional dose dependence of the CL inhibition for
BHT, we found that the concentration of 3.5 mg mL-1
was IC50. In addition, we found that A. onobrychis leaf
extract follows the BHT time course of CL prole. There
are some differences between crude and boiled plant
extracts. The crude extract shows approximately 27%
of CL inhibition, but the boiled extract inhibits 32% of
chemiluminescence (mean value). The inhibition index
is the same for different concentrations of leaf extract.
The plants have developed adaptations to extreme
climatic conditions: high temperatures, high light intensity
and irradiance, drought and freezing. Antioxidant defense
mechanisms can be vitally important in the survival of
wild plants [22,23]. LP is an indicator of the prevalence
of free radical reaction in tissues. Accumulation of O2•-
and • OH in the leaves contribute to lipid peroxidation
in A. onobrychis during the active vegetative period.
Even the level of LP did not signicantly change
during the examined vegetation period, as well as the
Table 3. Parameters of chemiluminescence inhibition by A. onobrychis leaf extracts.
Sample Percentage of inhibitionaIndex of inhibitionb
BHT (3.5 µg mL-1)49.1 -
BHT (7.0 µg mL-1)55.8 -
Crude leaves extract (3.5 µg mL-1)25.2 52.1
Crude leaves extract (7.0 µg mL-1)29.4 53.5
Boiled leaves extract (3.5 µg mL-1)30.1 62.1
Boiled leaves extract (7.0 µg mL-1)34.9 63.1
aCL sample/CL control × 100; bCL sample/CL BHT × 100
8
9
10
11
12
13
14
15
16
17
100 120 140 160 180 200 220 240 260 280 300 320 340
MDA (nmol mg protein)
-1
O·-(nmol mg protein)
2
-1
MDA = 7.39 + 0.02 ×O
·-
2
Correlation: r=0.82
95% confidence
8
9
10
11
12
13
14
15
16
17
1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3
MDA (nmol mg protein)
-1
·OH (nmol mg protein)
-1
MDA =-0.07 + 6.40 × ·OH
Correlation: r=0.70
95% confidence
AB
Figure 1. Evaluation of regression relationship in A. onobrychis leaf extract during the vegetative period considering: (A) quantities of MDA
and O2
•-; (B) quantities of MDA and •OH. Solid line is the tted linear regression line. Dashed lines represent the 95% condence limits.
4
5
6
7
8
9
10
11
12
100 120 140 160 180 200 220 240 260 280 300 320 340
Protein (mg g )
-1
O·-(nmol mg protein)
2
-1
Protein =11.93 - 0.02 ×O
·-
2
Correlation: r=-0.75
95% confidence
Figure 2. Evaluation of regression relationship between protein
content and quantities of O2
•- in A. onobrychis leaf
extract during the vegetative period. Solid line is the tted
linear regression line. Dashed lines represent the 95%
condence limits.
127

Antioxidative responses to seasonal changes and chemiluminescence
assay of
Astragalus onobrychis
leaves extract
•OH quantities, which indicates that the researched
plant species is not dramatically exposed to negative
inuences of ROS. As we pointed earlier, low level of
rainfalls during the vegetation period (Table 1), could
be the cause of drought, which is classied as an
extreme abiotic condition. This factor, which is probably
responsible for the signicant accumulation of O2•-,
was not destructive enough to cause the occurrence
of oxidative stress in the leaves of A. onobrychis.
The results obtained for ROS came from our earlier
research of Fabaceae plants [24]. The level of LP in
wild populations of Erica andevalensis, grown in metal-
enriched soils, was between 0.018 and 0.057 nmol mg-1.
These data suggest that E. andevalensis does not suffer
from oxidative stress derived from metal exposure and
accumulation [4]. The antioxidative protection in leaves
of Triticum aestivum varieties, with different eld drought
resistance, was studied under severe recoverable soil
drought at seeding stage by withholding irrigation for
7 days followed by re-watering. LP level was not changed
signicantly in the leaves of drought treated plants, but it
rose during recovery. These ndings indicate increased
oxidative strain on membranes in recovery from severe
drought stress but rather strict control on ROS formation
in the cells. The response of these varieties was similar
to conditions of drought and re-watering [25].
The soluble protein content is another indicator
of oxidative damage in a plant tissue. During growth,
protein content in leaves of A. onobrychis changed
signicantly and could be related to O2•- quantities. The
lowest protein content in leaves of examined plant was
in the seed forming stage. Similar results are published
for Astragalus mollissimus, in the seed stage of
vegetation, the protein content in leaves was the lowest
[26]. Different protein variations over seasons had been
observed in Picea omorika. Environmental parameters,
such as maximal and minimal temperatures, insolation,
wind power and frequency, peak in summer and winter,
and lower protein content was established [27].
The signicant accumulation of O2•- occurred in A.
onobrychis leaves, in the seed forming stage, should
go along with changes in SOD activity, but there is no
correlation of SOD activity and the O2•- quantities. It
can be assumed that the non-enzymatic components of
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0
8.2
8.4
8.6
8.8
45678910 11 12
POD (U mg protein)
-1
Protein (mg g)
-1
POD = 6.14 + 0.20 × Protein
Correlation: r=0.76
95% confidence
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
45678910 11 12
CAT(U mg protein)
-1
Protein (mg g)
-1
CAT=3.55 + 0.45 × Protein
Correlation: r=0.94
95% confidence
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0
8.2
8.4
8.6
8.8
100 120 140 160 180 200 220 240 260 280 300 320 340
POD (U mg protein)
-1
O·-(nmol mg protein)
2
-1
POD = 8.71 - 0.01 ×O
·-
2
Correlation: r=-0.74
95% confidence
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
100 120 140 160 180 200 220 240 260 280 300 320 340
CAT(U mg protein)
-1
O·-(nmol mg protein)
2
-1
CAT=8.99 - 0.01 ×O
·-
2
Correlation: r=-0.74
95% confidence
BA
DC
Figure 3. Evaluation of regression relationship in A. onobrychis leaf extract during the vegetative period considering: (A) CAT activities and protein
content; (B) POD activities and protein content; (C) CAT activities and quantities of O2
•-; (D) POD activities and quantities of O2
•-.
Solid line is the tted linear regression line. Dashed lines represent the 95% condence limits.
128

D. L. Miladinović et al.
antioxidant systems take over the role of O2•- scavengers.
It is also possible that the amount of superoxide anion
radicals was not large and that the activity of SOD was
quite optimal for scavenging O2•- to H2O2. Öncel et al.
[28] showed that both alpine and steppe plants may not
need all the components of antioxidant protection. SOD
activities in leaves of Astragalus vulneria and Astragalus
microcephalus were 60 and 127 unit g–1 FW. Anderson
et al. [29] in their study of seasonal variation in the
antioxidant system in Pinus strobus needles, showed
a minimal SOD activity in summer, and proposed that
maximal activity in winter might be a response to the
cold winter conditions. Salt stress treatment led to a
decrease of SOD activity in the seeds of Astragalus
adsurgens. The average values of SOD activities were
3.4 U mg-1 protein. The value of SOD activity of control was
5.0 U mg-1 protein [30]. SOD only exhibits maximum
activity in the seed forming stage when determining the
largest O2•- accumulation, which conrms its importance
as an indispensable component of the functioning
enzyme antioxidant system in wild plants.
The activity of SOD generates H2O2, while CAT
and POD are the main enzymes responsible for H2O2
decomposition thus preventing • OH generation [3].
Statistical tests conrms this fact. Change in CAT
activity during the examination vegetation period is
positively correlated with protein content. Catalase, in
joint action with guaiacol peroxidase, prevents •OH
formation, whose amount is not signicantly changed
during the season. These two enzyme systems are very
active and complement SOD activity. This assumption
can be indirectly derived on the basis of the statistical
test, namely POD and CAT activities were negatively
correlated with O2•- quantities. In an investigation of the
role of antioxidant defense system on stress tolerance
of high mountain and steppe plants, no CAT activity
was observed in steppe plants such as Astragalus
vulneria, Teucrium chamaedrys and Teucrium polium
[28]. In Kentucky bluegrass, the consistent and stable
expressions of CAT activity may facilitate leaf cells in
scavenging H2O2 in an efcient way. The combined
action of CAT and SOD prevent the cellular damage
under unfavourable conditions like water stress [31].
A relationship between protecting enzyme activities
and osmoregulation among three genotypes of Radix
Astragali under water decient conditions [32] conrms
that changes of SOD, CAT and POD activities are similar,
which indicated that these three enzymes cooperated
with each other. In the research of changes of antioxidant
enzymes activity in Astragalus membranaceus under
water stress, Kai et al. [33] found that POD activity initially
increased slowly but rose sharply in the later stage of
the stress under medium and severe drought. In the
literature data, there are signicant differences in POD
activity in some plant species. The reason for that are
several isoenzymes, which have separated physiological
functions. For example, in Triticum aestivum, exposure
to drought conditions, after rehydration, reveals three
isoforms of peroxidase [25].
Glutathione peroxidases were initially shown
to catalyze the glutathione dependent reduction of
hydrogen peroxide and diverse alkylhydroperoxides to
water or the corresponding alcohol via a thiol/disulde
exchange mechanism [34]. It has been found that plant
GPOX protective role arises during environmental
stresses and pathogen attack. However, plant GPOXs
have lower activities than those of animals because
they contain Cys at the putative catalytic site rather
than selenocysteine, typical of animal GPOXs. This low
01224364860728496 108 120 132
10
20
30
40
50
60
CL intensity (10 cpm)
3
time (min)
Control
BHT (3.5 µg mL )
-1
Crude extract (3.5 µg mL )
-1
Boiled extract (3.5 µg mL )
-1
Figure 4. Inhibition of chemiluminescence by A. onobrychis leaf
extract in lower doses.
01224364860728496 108 120 132
10
20
30
40
50
60
CL intensity (10 cpm)
3
time (min)
Control
BHT (7.0 µg mL )
-1
Crude extract (7.0 µg mL )
-1
Boiled extract (7.0 µg mL )
-1
Figure 5. Inhibition of chemiluminescence by A. onobrychis leaf
extract in higher doses.
129

Antioxidative responses to seasonal changes and chemiluminescence
assay of
Astragalus onobrychis
leaves extract
activity has made it difcult to clarify the physiological role
of GPOX in higher plants [35]. GPOX activity recorded
in A. onobrychis leaves extract was smaller, compared
with the results of published papers [36]. Based on the
results of our study, it can be assumed that GPOX from
the A. onobrychis leaves was included in the maximum
response to oxidative stress in the blooming vegetation
stage, under conditions of high light intensity and the
highest temperatures.
Methods of chemiluminescence are more sensitive
among assays for antioxidant activity estimation
because it detects the free radical trapping activity in
a chain reaction that occurs both in the hydrophilic and
the hydrophobic domains of biological membranes. The
luminol, as a chemiluminegenic probe, is very suitable
because its molecular weight is relatively small and
can enter cells that then react with intracellular ROS.
A kinetic prole of luminol-enhanced CL is dependent
on the type of activator. Zymosan activates an oxidative
burst of phagocytes by binding itself to complement
and immunoglobulin receptors, which induces signal
transduction that leads to the activation of protein kinase
C and a consequent activation of NADPH-oxidase, the
key enzyme of oxidative burst. The authors have chosen
BHT as an antioxidant standard because the long
lasting CL inhibition takes place, which is more suitable
in experiments on phagocytes [37].
The A. onobrychis extracts in both concentrations
were able to reduce emission of CL during the
metabolic phase of phagocytosis in a dose-dependent
manner. In our recent paper, CL inhibition by O. pilosa
extracts was observed on 33% and 35% (mean value)
of extracts. This can be explained by the fact that the
content of avonoids in leaves of O. pilosa is higher
than in A. onobrychis [8,24]. A study by Jantan et al.
[17] showed that most researched plant extracts did
not show signicant CL inhibitory effect in a similar
model system, except for Curcuma xanthorrhiza and
Garcinia mangostana. Compared to the common
antioxidant BHT, the inhibition of CL by A. onobrychis
extracts was lower, but compared to commercial vegetal
extracts Isoavin Beta and red clover, the extracts of A.
onobrychis demonstrated higher level of CL inhibition
[38]. Kawagoe and Nakagawa [39] have shown that, in
in vitro conditions, quercetin is a better suppressor of CL
intensity than the synthetic antioxidant BHT, in a dose-
dependent manner. In the study of antioxidant activity
determination of Astragalus squarrosus [40], it was
concluded that synergistic effects of different compounds
existing in the methanol extract of researched plant
might be responsible for their activities against lipid
peroxidation. However, the authors assume that the
weak free radical scavenging activity, observed in the
aerial part of the plant, might be related to the lack of
polysaccharides whose concentration is highest at the
root. Many studies have suggested that the presence
or position of specic glycoside groups can increase or
decrease the antioxidant activity of avonoids [41].
As mentioned earlier, in order to estimate
antioxidants of A. onobrychis by CL assay, two ways of
extract preparation were used: crude leaf extract and
boiled leaf extract (with denatured enzymes). The lower
inhibition of CL intensity was observed in the crude leaf
extract. The antioxidants present in crude leaf extract
may not be implicated in the main pathway, which leads
to a decrease of CL intensity. This is the case for some
enzymatic antioxidants such as SOD and CAT, which
were shown to be unable to affect chemiluminescence
at higher concentrations. The opposite effects could
involve direct stimulating action on the phagocyte cell,
chemical pro-oxidant action, and anti-inhibitory action
in different ways [42]. However, analyzing the results of
enzymatic activity (Table 2) and results of CL inhibition
of crude extracts (Table 3), it can be concluded that the
enzymatic compounds in the leaves of A. onobrychis
exhibit signicant antioxidant capacity, which is 50% of
CL inhibition activity of BHT. This activity protects plants
under climatic conditions with highest light intensities
and highest temperatures. Flavonoids, a major class
of antioxidant compounds, encompass a substantial
molecular weight range, which provides them with
different water solubilities [8]. The cell nucleus is an
aqueous domain; therefore, antioxidants which are
to act there should be water soluble. Flavonoids have
been shown to exhibit their antioxidant actions through
effects on membrane permeability, and by inhibition
of membrane-bound enzymes such as ATPase
and phospholipase [43]. In our study, based on the
percentage and index of inhibition (Table 3), boiled
extracts in which avonoids are the most represented
compounds, demonstrate a highest antioxidant activity.
In addition to ecological and botanical aspects, the
study of plant extracts can help in the design of modern
herbal medicinal products. It is important to note that the
synergistic, additive or potentiated effects shown by the
plant extract, frequently observed in the study of natural
products, usually exceed the effects of single compounds
or mixtures of them at equivalent concentrations [44].
4. Conclusions
The results presented suggest that the researched
antioxidant enzymes SOD, CAT, POD and GPOX
during vegetation signicantly change the levels of
activity in a specic way. During the spring season, POD
130

D. L. Miladinović et al.
showed the highest level. CAT and GPOX increased
their activities in summer. However, during the autumn
season, SOD showed maximum activity. This suggests
a complementary action of these enzymes in response
to external changes. Examined antioxidant enzymes
may be used as indicators of antioxidant ability of
A. onobrychis to environmental changes. Based on
CL activity, it can be concluded that the leaf extract of
A. onobrychis possesses a signicant antioxidant
capacity thus protecting plants during environmental
stress. Enzymatic components of A. onobrychis
antioxidant system, during the vegetative period, work
quite properly and the determined quantities of ROS are
not able to damage leaf cell structures.
Acknowledgements
We thank Dr. Novica Ranđelović for botanical determination
of plant species. This research was partially supported
by the Ministry of Education and Science of the Republic
of Serbia (Grant No. ON 171025)
A. Pitzschke, C. Forzani, H. Hirt, Antioxid. Redox
Sign. 8, 1757 (2006)
O. Ozkur, F. Ozdemir, M. Bor, I. Turkan, Environ.
Exp. Bot. 66, 487 (2009)
Z. Noreen, M. Ashraf, Environ. Exp. Bot. 67, 395
(2009)
B. Márquez-García, F. Córdoba, Environ. Exp. Bot.
68, 58 (2010)
D. Podlech, Proc. Roy. Soc. Edinb. B. 89, 37
(1986)
S.D. Lindblom et al., Environ. Exp. Bot. (in press),
DOI: 10.1016/j.envexpbot.2011.12.011
N. Diklić, In: M. Josifović (Ed.), Flora of Serbia
(Serbian academy of Science and Art, Belgrade,
1974) 274
D.L. Miladinović, B.S. Ilić, V.N. Milosavljević, Hem.
Ind. 65, 323 (2011)
A. Kabata-Pendias, Trace elements in soils and
plants, 4th edition (CRC Press, New York, 2011)
Z.A. Placer, L.L. Cushman, B.C. Johnson, Anal.
Biochem. 16, 359 (1966)
H.P. Misra, I. Fridovich, J. Biol. Chem. 247, 3170
(1972)
K.H. Cheeseman, A. Beavis, H. Esterbauer,
Biochem. J. 252, 649 (1988)
L.M. Simon, Z. Fatrai, D.E. Jonas, B. Matkovics,
Biochem. Physiol. Panzen 166, 387 (1974)
B. Matkovics, R. Novak, H.D. Hanh, Comp.
Biochem. Phys. 56, 397 (1977)
D.T.Y. Chiu, F.H. Stults, A.L. Tappel, Biochim.
Biophys. Acta 445, 558 (1976)
M.M. Bradford, Anal. Biochem. 72, 248 (1976)
I. Jantan, N. Hikmah Harun, A. Wira Septama,
S. Murad, M.A. Mesaik, J. Nat. Med. 65, 400
(2011)
W.S. Koko, M.A. Mesaik, S. Yousaf, M. Galal,
M.I. Choudhary, J. Ethnopharmacol. 118, 26 (2008)
V. Katsares et al., Lab. Med. 40, 557 (2009)
D. Miladinović, Antioxidant system of plants
(University of Niš, Niš, 2011)
S. Najman, Č. Radojičić, A. Dujić, Period. Biol. 88,
256 (1986)
R. Khanna-Chopra, D.S. Selote, Environ. Exp. Bot.
60, 276 (2007)
Y. Liang et al., Environ. Exp. Bot. 64, 286 (2008)
D. Miladinović, L. Miladinović, S. Najman, J. Serb.
Chem. Soc. 76, 505 (2011)
L. Simova-Stoilova, K. Demirevska, T. Petrova,
N. Tsenov, U. Feller, Plant Soil Environ. 54, 529
(2008)
P.A. Leand, MSc thesis, Texas Tech University
(Lubbock, USA, 1986)
J. Bogdanović, N. Milosavić, R. Prodanović,
T. Dučić, K. Radotić, Biochem. Syst. Ecol. 35, 263
(2007)
I. Öncel, E. Yurdakulol, Y. Keleş, L. Kurt, A. Yildiz,
Acta Oecol. 26, 211 (2004)
J.V. Anderson, B.I. Chevone, J.L. Hess, Plant
Physiol. 98, 501 (1992)
X. Wang, G. Zhao, H. Gu, Afr. J. Biotechnol. 8, 5773
(2009)
S.M. Bian, Y.W. Jiang, Sci. Hortic. 120, 264 (2009)
Y. Tan, Z. Liang, H. Shao, F. Du, Colloid. Surface.
B. 49, 60 (2006)
H. Kai, L. Zongsuo, D. Qimei, Acta Agr. Bor.-
Occident. Sinica 5, 100 (2010)
N. Navrot et al., Plant Physiol. 142, 1364 (2006)
Y. Miao et al., Plant Cell 18, 2749 (2006)
D.I. Štajner, B.M. Popović, P. Boža, A. Kapor,
Phytother. Res. 23, 1006 (2009)
L. Saleh, C. Plieth, Plant Methods 5, 1 (2009)
S.R. Georgetti, R. Casagrande, V.M. Di Mambro,
A.E.C.S. Azzolini, M.J.V. Fonseca, AAPS PharmSci
5, 1 (2003)
M. Kawagoe, K. Nakagawa, Toxicol. Lett. 114, 189
(2000)
J. Asgarpanah, S.M. Motamed, A. Farzaneh,
B. Ghanizadeh, S. Tomraee, Afr. J. Biotechnol. 10,
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
References
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
131

Antioxidative responses to seasonal changes and chemiluminescence
assay of
Astragalus onobrychis
leaves extract
19176 (2011)
J.G. Souza et al., Z. Naturforsch. C. 62, 339
(2007)
B. Gonzalez Flecha, S. Llesuy, A. Boveris,
Free Radical Bio. Med. 10, 93 (1991)
C. Desmarchelier, J. Coussio, G. Ciccia,
Braz. J. Med. Biol. Res. 31, 1163 (1998)
D.L. Miladinović et al., Anal. Bioanal. Chem. 403,
1007 (2012)
[41]
[42]
[43]
[44]
132