Advances in Environmental Biology Glycine max

Advances in Environmental Biology, 8(24) December 2014, Pages: 207-214
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Changes of Phytochemical Parameters and Antioxidant Enzymes in Glycine
max Under Pb Stress
1Behnaz
1
2
Kazemi, 2Leila Amjad, 2Monireh Ranjbar
Falavarjan Branch, Islamic Azad University, Isfahan, Iran.
Department of Biology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran.
ARTICLE INFO
Article history:
Received 26 September 2014
Received in revised form 25 November
2014
Accepted 21 December 2014
Available online 23 January 2015
Keywords:
Enzymatic antioxidants, Glycine max,
Lead, Phenoilc compounds.
ABSTRACT
Heavy metals modify growth, morphology and metabolism of plants in several ways.
To reveal metal toxicity, the effects of heavy metal Pb on phytochemical parameters of
Glycine max was determined. So, plants were treated with different concentrations (0,
5, 10 µM) of Pb and the contents of phenolic compounds, total flavonoid, flavonol ,
antioxidant activity and the activities of enzymatic antioxidants (peroxidase,
polyphenoloxydase) as marker for oxidative stress were investigated. This study,
factorial experimental design was ordered in a completely randomized block design
with three replications. Survey results indicated that the amount of non-enzymatic
antioxidant compounds (phenol, flavonoid and flavonol) and antioxidant activity
significantly were decreased in all treatments but a non-significant increase was
observed in polyphenol oxidase activity, whereas peroxidase was showed a significant
decrease. Although, increase in polyphenol oxidase activity is recognized as primary
response to metal toxicity for accumulation and tolerance to excess amount of heavy
metal, but this increase was not significant. These results show that different response
in control and treated plants is due to induction oxidative stress and heavy metal
toxicity in plants.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Behnaz Kazemi, Leila Amjad, Monireh Ranjbar., Changes of Phytochemical Parameters and Antioxidant Enzymes in
Glycine max Under Pb Stress. Adv. Environ. Biol., 8(24), 207-214, 2014
INTRODUCTION
Heavy metals are determined as that group of elements that have special weights higher than about 5g/cm 3.
A number of them (Fe, Co, Mn, Mo, Ni, Cu, Zn) are essential micronutrients and are required for growth and
take part in redox reactions, electron transfers and other important metabolic processes in plants. Metals which
are considered nonessential (Pb, Cd, Cr, Hg etc.) are potentially extremely toxic for plants [26]. Lead is known
to have toxic effects on membrane structure and functions [9]. Lead potentially induces oxidative stress and
evidence is storing to support the role of oxidative stress in the pathophysiology of lead toxicity [10].
Recent epidemiological studies have reported that low level lead exposure has a graded association with
several disease consequences such as kidney disease, hypertension, peripheral artery disease, neurodegenerative
disease and cognitive impairment. Although all these diseases include components of oxidative stress, the
relationship of oxidative stress to lead-related disease with low level exposure has been dispraised because
studies have been conducted at levels not typically observed in common population [22].
Both abiotic and biotic stresses are known to involve plants to produce reactive oxygen species (ROS) that
can cause damage to the tissues or signal the start of physiological defence reactions [7]. Usually, as a result of
various biotic and abiotic stresses, increased L-phenylalanine ammonialyase activity and accumulation of many
phenolics are observed [30,14]. In abiotic sources, plants produce ROS by primary metabolic processes such as
chloroplastic and mitochondrial electron transport [11].
In addition, plants synthesise compounds which are able to reduce the damaging effects of different
stresses. The antioxidant properties of phenolics are well-documented [13]. These compounds are present in
plants as fundamental or can be synthesised de novo [15]. It is known that the effect of phenolics on growth is a
complex process. These compounds may interfere in auxin metabolism, change membrane penetrance, influence
respiration and oxidative phosphorylation or protein synthesis [4]. The phenolic compounds such as flavonoids
Corresponding Author: Behnaz Kazemi, Falavarjan Branch, Islamic Azad University, Isfahan, Iran.
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Advances in Environmental Biology, 8(24) December 2014, Pages: 207-214
are active antioxidants [25] and also cause appeasement of lipid peroxidation [32]. Plants possess enzymatic
systems that protect them against H2O2 and other harmful ROS. These include superoxide dismutase, catalase,
peroxidase, polyphenol oxidase etc. [33].
Therefore, the present study was to evaluate: the effects of different concentrations of Pb on the
phytochemical and activity of enzymatic antioxidants (peroxidase, polyphenol oxidase) on Pb stress.
MATERIALS AND METHODS
Plant materials:
Soybean seeds (Glycine max var.Williams) were supplied from Oil Seeds Center (Isfahan, Iran). The seeds
were saturated with 2-3% Hydrochloric acid solution and were soaked in distilled water for 24 h and then were
germinated in pots containing perlite and coco peat (1 seed per pot) and watered with half-strength Hogland
nutrient solution. The plants were grown at 25°C temperature, with a 16/8 h day/night photoperiod for 20 days.
Seedling at the three-leaf stage treated at different concentrations of Pb(NO3)2 (0, 5, 10 µM). 20 days after
treatment, plants were removed and they were washed with distilled water and separated from the aerial parts.
Ten grams of each plant powder was extracted in 150 ml of 80% methanol by maceration (72 h). The solvent
was removed under the vacuum at temperature below 50 oC and the extracts were freeze-dried. Experiments
were conducted at Botany lab, Department of Biology, Faculty of Biological Sciences, Islamic Azad University
of Falavarjan, Isfahan, Iran.
Total phenol determination:
Total phenols were determined by Folin Ciocalteu reagent [29,31]. Different concentrations of each plant
extract (10, 20, 40, 60, 80, 100 µl) or gallic acid (standard phenolic compound) was mixed with Folin Ciocalteu
reagent (1 ml, 1:10 diluted with distilled water) and 7% Na2CO3 (1 ml). The mixtures were allowed to stand for
15 min and the total phenols were determined by colorimetry at 765 nm. The standard curve was prepared using
0, 50, 100, 150, 200, 250 mg L-1 solutions of gallic acid in methanol : water (50:50, v/v). Total phenol values are
expressed in terms of gallic acid equivalent (mg g –1 of dry mass), which is a common reference compound.
Total flavonoid and flavonol determination:
Aluminum chloride colorimetric method was used for flavonoids determination [21,24]. Each plant extracts
(10, 20, 40, 60, 80, 100 µl) in methanol were separately mixed with 1.5 ml of methanol, 1 ml of 2% aluminum
chloride, 6 ml of 5% potassium acetate and 2.8 ml of distilled water. It remained at room temperature for 40
min; the absorbance of the reaction mixture was measured at 415 nm for flavonoid assay and then was measured
at 440 nm for flavonol assay. The calibration curve was prepared by preparing rutin solutions at concentrations
12.5 to 100 g ml-1 in methanol.
Free radical scavenging activity determination:
The stable 1,1-diphenyl-2-picryl hydrazyl radical (DPPH) was used for determination of free radicalscavenging activity of the extracts [16]. Different concentrations of each extract (10, 20, 40, 60, 80, 100 µl)
were added, at an equal volume, to methanolic solution of DPPH (0.004g per 100 ml) . After 120 min at room
temperature, the absorbance was recorded at 517 nm. The experiment was repeated for three times. Ascorbic
acid were used as standard controls. IC50 values denote the concentration of sample, which is required to
scavenge 50% of DPPH free radicals.
Enzyme extraction and assay:
The samples, weighing about 50 mg, were homogenized with 2 ml of phosphate buffer pH 6.8 (0.1 M). This
portion was centrifuged at 4°C for 10 min at 15,000g in a refrigerated centrifuge. The clear supernatant was
taken as the enzyme source.
Peroxidase Assay:
2.7 ml sodium phosphate buffer (25 Mimoles), pH 6.8, 100µl gayagol (20 Mi moles), 100µl of H202(40
Mimoles), and 100 µl of the enzyme extract. The absorbance of the reaction mixture was measured at 470 nm at
time (15, 75 s) for peroxidase assay.
Polyphenoloxidase Assay:
2.8 ml sodium phosphate buffer( 25 Mimoles), pH 6.8, 100µl pirogalol(10 Mi moles) and 100 µl of the
enzyme extract. The absorbancy of the purpurogallin formed was taken at 420 nm at time (40, 100s) for
polyphenol oxidase assay (Kar and Mishra, 1976).
Statistical analysis:
The experimental design was a split plot in a randomized complete block design with three replications.
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The presented data included means of three separate experiments ± SD. In order to analyze the data, SPSS
software and ANOVA test were used. Thus, the statistical significance between phytochemical activities values
of the extracts was evaluated with a LSD test. P values less than 0.05 were considered to be statistically
significant.
Results:
Analysis of data on total phenol showed that Pb poisoning caused significant reduction in polyphenol
content compared to non-Pb treatment(P<0.05), so that when Pb concentration was increased, the amount of
polyphenol content was declined (Figure 1).
0.700
0.600
OD
0.500
0.400
0.300
0.200
0.100
0.000
0.01
0.02
0.04
0.06
Concentration(mg/ml
Non-pb
5 µmol pb
0.08
0.1
)
10 µmol pb
Fig. 1: Significant reduction of phenol content (concentration and absorbance) of under treatment groups with
Pb 5 and 10µM in compared with non-Pb group. Bars are least significant differences where p < 0.05.
Table 1 show the content of total phenols that were measured by Folin Ciocalteu reagent in terms of gallic
acid equivalent (standard curve equation: y = 29.85x +0.043, r 2= 0.990). The total phenol varied from 38.86 ±
0.05 to 166.83 ± 0.02 mg g-1 in the extract powder. Non-Pb group with total phenol content of 166.83±0.02 mg
g-1 had the highest amount among the plants in this study (Figure 2).
Total phenol(mg/g GA)
Table 1: Phenol content in the studied plant extracts.
1
Groups
Mean Total phenol ±SD
Pb-5µM group
42.88±0.2
Pb-10µM group
38.86±0.05
Non-Pb group
166.83±0.02
1
Each value in the table was obtained by calculating the average of three experiments ± standard deviation.
200
180
160
140
120
100
80
60
40
20
0
Non-pb
5 µmol pb
10 µmol pb
Sample
Fig. 2: Significant reduction of total phenol(mg/g GA) of under treatment groups with Pb 5 and 10µM in
compared with non-Pb group. Bars are least significant differences where p < 0.05.
When lead concentration was increased in a treatment, the flavonoid and flavonol content were significantly
decreased compared to control group (P<0.05). So that when Pb concentration was increased, the amount of
polyphenol content was declined, but this reduction was not significant (Figure 3,4).
The flavonoid content of the extracts in terms of rutin equivalent (the standard curve equation: y = 0.0603x +
0.0007, r2 = 0.985) were between 2.625 ± 0.001 and 5.975 ± 0.028 (Table 2). The flavonoid content in the
extracts of under treatment with Pb 5 µM (3.106 ± 0.013 mg g-1) and under treatment with Pb 10 µM (2.625 ±
0.001 mg g-1) were lower than that in the extract of Non-Pb(5.975 ± 0.028 mg g-1) (Figure 5). Table 2 also
show the content of total flavonol that were measured in terms of rutin equivalent (standard curve equation: y =
210
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Advances in Environmental Biology, 8(24) December 2014, Pages: 207-214
0.0603x + 0.0007, r2 = 0.985)). The total flavonol varied from 1.71 ± 0.03 to 3.30 ± 0.01 mg g-1 in the extract
powder. Non-Pb group with total flavonol content of 3.30±0.01 mg g-1 had the highest amount among the
plants in this study (Figure 6).
0.450
0.400
0.350
OD
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0.05
0.1
Non-pb
0.25
0.5
Concentration(mg/ml)
5 µmol pb
0.75
1
10 µmol pb
Fig. 3: Significant reduction of flavonoid content (concentration and absorbance) of under treatment groups
with Pb 5 and 10µM in compared with non-Pb group. Bars are least significant differences where p <
0.05.
0.25
0.2
OD
0.15
0.1
0.05
0
0.05
0.1
Non-pb
0.25
0.5
Concentration(mg/ml)
5 µmol pb
0.75
1
10 µmol pb
Fig. 4: Significant reduction of flavonol content (concentration and absorbance) of under treatment groups with
Pb 5 and 10µM in compared with non-Pb group. Bars are least significant differences where p < 0.05.
Total flavonoid (mg/g RTA)
Table 2: Flavonoid content in the studied plant extracts.
1
1
Groups
Mean Total flavonoid ±SD
Mean Total flavonol ±SD
Pb-5µM group
3.106±0.013
2.06±0.02
Pb-10µM group
2.625±0.001
1.71±0.03
Non-Pb group
5.975±0.028
3.30±0.01
1
Each value in the table was obtained by calculating the average of three experiments ± standard deviation
7
5.975
6
5
3.106
4
2.625
3
2
1
0
Non-pb
5 µmol pb
Sample
10 µmol pb
Fig. 5: Significant reduction of total flavonoid (mg/g RTA) of under treatment groups with Pb 5 and 10µM in
compared with non-Pb group. Bars are least significant differences where p < 0.05.
Figure 7 shows the amount of each extract needed for 50% inhibition (IC50). IC50 of the standard
compounds, Ascorbic acid were 58.85 µg/µl. The highest radical scavenging activity was showed by non-Pb
group with IC50=61.72 which is nearby of Ascorbic acid. The radical scavenging activity in the plant extracts
decreased in the following plants: under treatment groups with Pb 5 and 10µM(Figure 8).
Increasing concentrations of lead, significantly (P <0.05) decreased peroxidase enzyme activity (Figure 9),
whereas polyphenoloxidase enzyme activity under different lead treatments were increased, but this different
were not significant (Figure 10).
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Total flavonol (mg/g RTA)
Advances in Environmental Biology, 8(24) December 2014, Pages: 207-214
3.3
4
3.5
3
2.5
2
1.5
1
0.5
0
2.06
1.71
Non-pb
5 µmol pb
10 µmol pb
Sample
Fig. 6: Significant reduction of total flavonol (mg/g RTA) of under treatment groups with Pb 5 and 10µM in
compared with non-Pb group. Bars are least significant differences where p < 0.05.
IC50
95
85
IC 50(µg/µl)
75
65
[81.83]
[74.36]
55
[61.72]
45
35
25
Non-pb
5 µmol pb
10 µmol pb
Sample(%)
Fig. 7: IC50 (µg/µl-1) values of plant extracts for free radical scavenging activity by DPPH radical. Lower IC50
value indicates higher antioxidant activity.
0.600
0.500
OD
0.400
0.300
Non-pb
0.200
5 µmol pb
0.100
10 µmol pb
0.000
0.01
0.02
0.04
0.06
0.08
0.1
Concentration(mg/ml)
OD
Fig. 8: Significant increase of antioxidant activity (concentration and absorbance) of under treatment groups
with Pb 5 and 10µM in compared with non-Pb group. Bars are least significant differences where p <
0.05.
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
15
25
35
10 µmol
45
Time
5 µ mol
55
60
65
75
non-pb
Fig. 9: Significant reduction of peroxidase enzyme (time and absorbance) of under treatment groups with Pb 5
and 10µM in compared with non-Pb group. Bars are least significant differences where p < 0.05
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Behnaz Kazemi et al, 2014
OD
Advances in Environmental Biology, 8(24) December 2014, Pages: 207-214
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
10 µmol
5 µmol
40 second
non-pb
Sample
100 second
Fig. 10: Significant increase of polyphenol oxidase enzyme (time and absorbance) of under treatment groups
with Pb 5 and 10µM in compared with non-Pb group.
Discussion:
According to the results under different treatments of lead in the study, total phenol, flavonoid and flavonol
content, antioxidant activity and peroxidase enzyme activity sighnificantly decreased as compared to the
control(P<0.05). On the other hand, polyphenoloxidase enzyme activity were increased. This results were
similar with results of Yoshimurak et al. [35], they reported that high concentrations of heavy metals can
decrease activity of antioxidant enzymes.
Heavy metals Cd, Pb, Al, Zn, and Cu induce oxidative stress in plant species [6,20,28]. The most of Pb
toxicity in plants is retard growth and alteration in the activity of many key enzymes of various metabolic
pathways [23,1]. the mechanisms of ROS production by metals such as Pb, Cd, and Zn ions is less clear, and
perhaps mediated, for example, by activation of lipoxygenase or binding to membrane proteins, thus precipitate
electron leakages responsible for formation of ROS [27].
The antioxidant enzymes are main cellular defense system against oxidative stress [12,19]. Pb stress in the
present study resulted in a high activity of polyphenoloxidase in the plant, the high polyphenoloxidase activity
could possibly be the result of both a direct effect of Pb stress in plant tissues and an indirect effect mediated via
an increase in levels of O2 [6].
The peroxidase regulation as a response of the plant to pollutants can already be used for the
phytomonitoring of industrial or thickly urbanised areas. Peroxidases have been shown to be totally sensitive to
pollution [34].
Arora et al. [2] show that phenolics (especially flavonoids) are able to alter peroxidation kinetics by
changing the lipid packing order. They stabilize membranes by decreasing membrane fluidity and hinder the
spread of free radicals and limit peroxidative reaction [2,3].
Antioxidant activity of phenolic compounds is due to their high trend to chelate metals. Phenolics possess
hydroxyl and carboxyl groups, able to bind especially heavy metals [17].
Change in soluble phenolics such as mediator in lignin biosynthesis can reflect the typical anatomical
change induced by stressors: increase in cell wall tolerance and the creation of physical barriers preventing calls
against harmful activity of heavy metals [8]. In recent years there has been a growing interest in antioxidant
properties of phenolic compounds.
Conclusion:
Heavy metals, especially lead, are a toxic factor limiting crop production. The present study demonstrated
that in plants under treatment with Pb, amount of phenol, flavonoid and flavonol compounds and antioxidant
activity significantly were decreased but a non-significant increase was observed in polyphenol oxidase activity,
whereas peroxidase was showed a significant decrease. These changes were related to many factors including
metal concentrations, plant species and plant tissues. It has now become clear that high to moderate doses of
lead exposure cause generation of free radicals resulting in oxidative damage to final biomolecules, lipids,
proteins and DNA.
ACKNOWLEDGEMENT
This work was supported by Islamic Azad University, Falavarjan Branch; the authors also thank from
research assistant for their kindly aid.
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