Advances in Environmental Biology Antioxidant

Advances in Environmental Biology, 8(24) December 2014, Pages: 185-193
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Assessment of the Effect of Electromagnetic Fields on Biochemical and
Antioxidant Parameter Changes of Cucurbita maxima Duchesne.
Simin Nabizadeh, Ahmad Majd, Sadigheh Arbabiyan, Masoumeh Mirzai, Fariba Sharifnia
Department of Biology, Faculty of Biological Sciences, North-Tehran Branch, Islamic Azad University, Tehran, Iran.
ARTICLE INFO
Article history:
Received 26 September 2014
Received in revised form
20 November 2014
Accepted 25 December 2014
Available online 20 January 2015
Keywords:
Electromagnetic fields, antioxidant
activities, FRAP, DPPH, Cucurbita
maxima Duchesne.
ABSTRACT
Since electromagnetic. In fields as a environmental factor can affect living organisms
and cause oxidative stress, in the present study therefore, the effects of electromagnetic
field (2mT) on some antioxidant enzymes activities of Cucurbita maxima Duchesne
such as catalase, peroxidase and superoxide dismutase were assessed. Moreover, some
other biochemical parameters (FRAP and DPPH) under electromagnetic field stress
were studied. Wet and dry seeds of Cucurbita maxima Duchesne were exposed to the
2mT electromagnetic field, for 15 and 30 minutes. The fruits of mature Pumpkin, were
used as samples for following biochemical and antioxidant assessments .The results
indicated that the changes of some antioxidant activity were miscellaneous and
unexpected. So that, up to 30 minutes exposing to electromagnetic field, the protein
quantity was gradually decreased. Moreover, all dry treated seeds showed more
superoxide dismutase activity than wet treatments. 15min dry treatment significantly
increased catalase activity of samples, followed by control dry samples. In the
peroxidase case it was also indicated that samples of control dry treatment have the
highest activity. On the other hand, DPPH and FRAP parameters were significantly
increased in 30 and 15 min treatments, respectively ( both dry and wet seeds).
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Simin Nabizadeh, Ahmad Majd, Sadigheh Arbabiyan, Masoumeh Mirzai, Fariba Sharifnia., Assessment of the Effect
of Electromagnetic Fields on Biochemical and Antioxidant Parameter Changes of Cucurbita maxima Duchesne.. Adv. Environ. Biol.,
8(24), 185-193, 2014
INTRODUCTION
Recognition and responding to the environmental stresses, is one of the plant’s capability. the balance
between the production of reactive oxygen species (ROS) and the quenching activity of antioxidants, both are
upset when plants are subjected to environmental stress, and it often brings about oxidative damage ]3].
Although ROS are produced within cells as a consequence of normal metabolic processes, under stress
condition the production of ROS often increases [32]. ROS participate in signal transduction and modification
of cellular components, which cause damage. Abiotic stress results in the formation of ROS in plants that
makes a condition called oxidative stress[3].
Under stress condition, production of ROS is unavoidable and therefore plants need to have a set of
enzymatic and non-enzymatic antioxidant molecules to protect themselves from cellular damage produced by
ROS [3,5,9]. There is a possible relationship between abiotic stress such as electromagnetic field radiation
(EMFr) and its effects on living organisms which can bring about an oxidative stress. This stress in turn,
increases activity, concentration and lifetime of free radicals [2,17]. External magnetic fields are found to be
more efficacious than geo-magnetic field (GMF) on biological systems [37,14]. Many authors have reported that
the rate and percentage of seeds germination, seedlings development, reproduction and growth of meristem cells
, are affected by magnetic fields [4,18,26, 27, 14]. It is shown that in some cases, cause higher yield [23]. EMFs
increase percentage of germination in Cucumis sativus L. seeds [38]. On the other hand, it is reported that EMFs
decrease speed of germination in Vicia sativa L. [14]. EMFs can change the activity of some antioxidant
enzymes such as superoxide dismutase, ascorbate peroxidase ,glutathione reductase and catalase [12, 31]. It is
also reported that high electromagnetic field increase the amount of plastoglobules, and can change the structure
and export products of Golgi apparatus [25]. Lipid peroxidation and hydrogen peroxide content also affected by
EMFs [35]. As low frequency electromagnetic fields are environmental factors through out the earth [20] , the
Corresponding Author: Simin Nabizadeh, Department of Biology, Faculty of Biological Sciences, North-Tehran Branch,
Islamic Azad University, Tehran, Iran.
E-mail: [email protected]
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Advances in Environmental Biology, 8(24) December 2014, Pages: 185-193
aim of the present study therefore , is to assay The effect of electromagnetic fields on biochemical and
antioxidant parameter changes of Cucurbita maxima Duchesne. Cucurbita maxima Duchesne is a prostrate,
coarse, annual vine that grows to a length of 4 m or more. [19]. The fruits, flowers and young shoots of this
plant are used as vegetable. Moreover, the fruits of Pumpkin are good sources of calcium, phosphorus and iron
[15]. It has been reported that Cucurbita maxima has antidiabetic, hepatoprotective, anthelmintic,
antihypertensive, anticancer and antihypercholesterolemic features [29]. Moreover, the results of several studies
showed the remedial effect of pumpkin seed on prostatic hyperplasia [10, 11, 13]. Cucurbita maxima seed oil
has also been found to reduce the size of prostate [16].
METHOD AND MATERIALS
Electromagnetic field source:
The source of the magnetic field was means of a pair of Helmholtz coils system which creates a uniform
magnetic field into rather large space volume .Each Helmholtz coil from exposure device has a diameter by
260mm and 1000number of turns ( Fig 1).
Fig. 1: Source of Electromagnetic field.
Experiment implementation:
Seeds of Cucurbita maxima Duchesne were purveyed from Sina Bazr Alvand Co. There were two different
treatments based on the length of exposing samples to electromagnetic field, so that 15 and 30 min treatments,
by a magnitude of 2 mT for 5 days, in Petri dishes were accomplished. Moreover, there were two groups of
seeds. The first one which called dry, and the second one called wet seeds that were soaked for 24 h, before
being treated. For both wet and dry treatments, separate groups of control samples were considered , so that they
were placed in the similar coil which was disconnected to the power (Table1). For each treatment, there were at
least 6 replications.The temperature was adjusted to (24±0.5c). Photoperiods was 14 light/10 darkness, (10-13
MJ m-2 d-1). After treatments, the seeds were allowed to germinate and then, transferred to jardinières. After 70
days, when the fruits of Cucurbita maxima Duchesne, including all samples of the tretments were formed and
maturated, tissue of fruits were collected for following experiments.
Table 1: Treatments configuration.
Exposure Time ( min)
15
30
0 ( control)
State seed
Wet
Dry
Wet
Dry
Wet
Dry
Electromagnetic treatment(mT)
2mT
2mT
0 ( control)
Protein extraction:
In order to assaying of protein content and catalase, Peroxidase and superoxid dismutase activities, fruits
tissue of each treated and control samples (0.15 mg) was ground in 5 ml of 100 M phosphate buffer (pH 7)
under ice-cold condition and then was centrifuged at 12000 rpm at 4´C for 45 min, separately.
Protein content assay:
Bradford's method (1976) was used for comparison of total protein content. Bovine serum albumin (BSA)
was used as reference standard. 90 μl phosphate buffer and 5 ml Bradford's reagent was added to 10 μl protein
extraction and after 5 min, the absorbance was measured at 595 nm. The protein concentration was expressed as
mg ml-1.
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Catalase assay:
Reaction mixture (3 ml) included 50mM potassium phosphate buffer (pH 7), 20 mM Hydrogen peroxide
and 100 μl enzyme extraction. CAT activity was determined as the rate of disappearance of H2O2 at 240 nm,
according to Pereira. The activity of catalase was expressed as μmol -1min-1mg protein.
Peroxidase assay:
The activity of POD was measured according to the method of Biles and Abeles. The 4 ml reaction mixture
contained 0.2 M (pH 5) Acetate buffer, 400 μl of 3% H2O2, 200 μl of 0.02 M benzydin, 50 ml of 50% methanol
and 100 μl of enzymes extract. The change in absorbance was determined within 1 min at 590 nm. The activity
of Peroxidase was expressed as μmol-1min-1mg protein.
Superoxide dismutase assay:
The SOD activity inhibits the photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm. The
monitoring of this inhibition is used to assay SOD activity. The reaction mixture was prepared by taking 50 μL
enzyme extract and adding 1 mL NBT (50 μM), 500 μL methionine (13 mM), 1mL riboflavin (1.3 μM), 950 μL
(50 mM) phosphate buffer and 500 μL EDTA (75 mM). This reaction was started by keeping reaction solution
under 30 W fluorescent lamp illuminations and turning the fluorescent lamp on. The reaction stopped when the
lamp turned off 5 min later. The NBT photo reduction produced blue formazane which was used to measure the
increase in absorbance at 560 nm. The activity of SOD was expressed as μmol-1min-1mg protein.
DPPH and FRAP assay:
For the determination of DPPH (1, 1-diphenyl-2-pycrylhydrazyl) radical scavenging, 1 ml of methanol
extracts of fruit was mixed with 5 ml of 0.04% (w/v) DPPH in methanol, mixed properly and the reaction
mixture was kept in dark at room temperature for half an hour. The absorbance of mixture was then measured at
517 nm using spectrophotometer and the results were expressed in µM TE/g fresh mass (Liyana-Pathirama et al
,2005). FRAP(ferric reducing antioxidant power) assay was accomplished by adding 10 ml of 300 mM acetate
buffer (3.1g of sodium acetate and 16 ml glacial acetic acid per liter), 1ml of 10 mM 2,4,6-tri-2pyridyl-1,3,5triazine (TPTZ) in 40 mM HCl and 1 ml of 20 mM ferric chloride. The mixture was pre warmed at 350´C. Three
ml of mixture was added to 1ml of extract and kept at room temperature for 10 min. The absorbance of resulting
mixture was read at 593 nm and the results were expressed in µM TE/g fresh mass.
Statistical Analysis:
SPSS ver16. was used for statistical analysis. Means were compared using the duncan test at P<0/05, level
of significance to distinguish the differences between treatments and control samples. There were three
replicates for all experiments. data were expressed as the mean ± SE. The graphs were plotted using Excel
software.
Results:
Protein content:
The assessment of total protein content showed that control samples of wet-treated seeds of Pumpkin, have
the highest protein proportion, followed by 15 min wet-treated samples (2mT). However, 30 min wet-treated
samples contain the lowest total protein content. There were no significant differences between 15min and
30min dry samples (Table 2, Figure2).
Catalase activity:
15min treatment of electromagnetic field (2mT) on dry seeds of Pumpkin, significantly and highly
increased the activity of catalase, in comparison to other treatments. Here , no significant differences detected
between 15 min and 30 min wet-treated samples. The lowest value of catalase activity belonged to 30 min drytreatment (Table2, Figure3).
Peroxidase activity:
As shown in table 2 and figure 4, the activity of peroxidase in control dry samples of Pumpkin, is
significantly higher than other treatments.The samples of 15min dry-treated seeds (2mT) also have high
peroxidase activity. On the other hand, control samples of wet-treated seeds, were found to be at the lowest level
of peroxidase activity. No significant differences were detected among 15min and 30 min wet- treated and
30min dry-treated samples, in terms of peroxidase activity.
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Table 2: The effect of electromagnetic field (2mT) on protein content and antioxidant activities and parameters of Cucurbita maxima
Duchesne. Means ± SE and P ≤ 0.05. The letters show significance of differences.(D: dry seeds, W : wet seeds).
Protein content
Catalase
Peroxidase
Superoxide
FRAP
DPPH
mg ml-1
activity
activity
dismutase activity
µM TE/g fresh
µM TE/g fresh
μmol-1min-1mg
μmol-1min-1mg
μmol-1min-1mg
mass
mass
protein
protein
protein
control D
control
W
15min D
30min D
15min W
30min W
0.184±SE
c
0.21±SE
a
0.165±SE
d
0.164±SE
d
0.2±SE
b
0.16±SE
e
0.055±SE
b
0.02±SE
c
0.088±SE
a
0.01±SE
e
0.013±SE
d
0.013±SE
d
0.34±SE
a
0.026±SE
d
0.169±SE
b
0.043±SE
c
0.036±SE
c
0.045±SE
c
0.442±SE
a
0.175±SE
d
0.242±SE
c
0.371±SE
b
0.096±SE
e
0.06±SE
f
775.25±SE
d
834.18±SE
c
893.12±SE
b
539.45±SE
f
1148.53±SE
a
735.95±SE
e
5.51±SE
cd
6.21±SE
c
4.61±SE
de
10.58±SE
a
4.48±SE
e
7.54±SE
b
Protein
0.25
a
c
0.2
b
d
d
e
0.15
Mg ml-1
0.1
Prot…
0.05
0
Control D Control W 15minD
30minD
15minW
30minW
Treatments
Fig. 2: The effect of electromagnetic field (2mT) on protein content of Cucurbita maxima
Duchesne. The letters
show significance of differences.(D: dry seeds, W : wet seeds).
C atalas e
-
μmol
-1
min mg
protein
a
b
c
e
d
d
C atalas e
on
C
C
on
tro
lD
tro
l
15 W
m
i
3 0 nD
m
1 5 inD
m
i
3 0 nW
m
in
W
1
0.1
0.08
0.06
0.04
0.02
0
Treatments
Fig. 3: The effect of electromagnetic field (2mT) on catalase activity of Cucurbita maxima Duchesne. The
letters show significance of differences.(D: dry seeds, W : wet seeds).
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P eroxidas e
0.4
0.3
0.2
0.1
0
a
b
c
d
c
c
P erox idas e
Co
n
C o tro
nt l D
ro
15 l W
m
30 inD
m
15 in
m D
30 inW
m
in
W
μm ol 1
m in -1 m g
prote in
Treatments
Fig. 4: The effect of electromagnetic field (2mT) on peroxidase activity of Cucurbita maxima Duchesne. The letters show
significance of differences.(D: dry seeds, W : wet seeds).
Superoxide dismutase activity:
Just like peroxidase, the activity of superoxide dismutase was also found to be at the highest level in
control dry samples. It is followed by samples of 30min dry-treated, 15min dry-treated, wet control and 15min
wet-treated seeds, respectively. 30min wet-treated samples showed the lowest level of superoxide dismutase
activity (Table2, Figure5).
S uperoxid
a
b
d
c
e
f
S uperox id
Co
n
C o tro
nt l D
ro
15 l W
m
3 0 in D
m
1 5 in D
m
30 inW
m
in
W
μm ol 1
m in -1 m g
prote in
0.5
0.4
0.3
0.2
0.1
0
Treatments
Fig. 5: The effect of electromagnetic field (2mT) on superoxide dismutase activity of Cucurbita maxima Duchesne. The
letters show significance of differences.(D: dry seeds, W : wet seeds).
FRAP (ferric reducing antioxidant power):
Samples of both 15min wet and dry treated seeds of Pumpkin, showed the highest ferric reducing
antioxidant power, respectively. There are significant differences among other treatments and the lowest ferric
reducing antioxidant power was found in 30min wet-treated samples (Table2, Figure6).
Radical scavenging of DPPH:
Despite PRAP, the 1,1-diphenyl-2-pycrylhydrazyl radical scavenging, as shown in table 2 and figure7, has
the highest level in both 30min wet and dry treatments. The results indicated that 15min wet and dry treatments
have the lowest level of DPPH radical scavenging.
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FRAP
1400
1200
1000
800
μM TE/g
fresh mass 600
400
200
0
a
b
c
d
e
f
FRAP
Treatments
Fig. 6: The effect of electromagnetic field (2mT) on FRAP of Cucurbita maxima Duchesne. The letters show significance of
differences.(D: dry seeds, W : wet seeds).
DP P H
de
e
W
m
30
in
m
15
in
W
D
in
30
m
in
m
15
W
tro
l
C
on
D
DP P
H
tro
l
on
C
b
c
cd
D
μM T E /g fres h
mas s
a
12
10
8
6
4
2
0
Treatments
Fig. 7: The effect of electromagnetic field (2mT) on DPPH radical scavenging of Cucurbita maxima Duchesne.
The letters show significance of differences.(D: dry seeds, W : wet seeds).
Discussion:
In the present study, the activity of some antioxidant enzymes and biochemical parameters, as well as total
protein content of Cucurbita maxima Duchesne were assessed, under electromagnetic field stress. Fruit tissue of
Pumpkin which their seeds had been already treated by 2mT electromagnetic field was used as samples.
Although DPPH and FRAP parameters were significantly increased in 30 and 15 min treatments respectively
(both dry and wet seeds) , the activity of some antioxidant enzymes undergo miscellaneous and somewhat
unexpected changes. In the FRAP case, it seems that up until 15 min exposing to the electromagnetic field,
ferric reducing antioxidant power was increased gradually. But after the critical point which might be more or
less 15 minutes, this parameter (FRAP) dwindles up to 30 minutes. On the other hand, increasing of DPPH
radical scavenging follows the different pattern, so that 30min treatments (wet and dry) and 15min treatments
(wet and dry) found to be at the highest and lowest level of DPPH radical scavenging, respectively. It means
that being wet or dry seeds, during electromagnetic field treatments, does not have remarkable effects on the
following DPPH radical scavenging value. Total protein content assessment showed that up to 30 minutes
exposing to electromagnetic field, the protein quantity was gradually decreased.
The pattern of superoxide dismutase changes was different. All dry treated seeds showed more superoxide
dismutase activity than wet treatments. However, control dry treatment interestingly found to be at the highest
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level of superoxide dismutase activity followed by 30min dry treatment. In the peroxidase case it was also
indicated that samples of control dry treatment have the highest activity, followed by 15min dry treatment.
15min dry treatment significantly increased catalase activity of samples, followed by control dry samples.
Here again it can be concluded that 15 minutes treatment of electromagnetic field (2mT), is the critical point of
catalase activities.In the previous studies, Farzpourmachiani. S et al reported that electromagnetic fields
decreased protein content and increased catalase activity of Valerian leaves, specially in dry treated seeds [8].
These results are similar to that of indicated in the present study. On the other hand, it is shown that protein
content and catalase activity of soybean were increased by pulsed magnetic field [21]. Shabrangi.A et al
showed that catalase and superoxide dismutase activities of Zea mays L. [28]. were increased by
electromagnetic field treatment. Although the catalase activity in the present study also was increased, the
activity of superoxide dismutase showed different pattern of changes. They also reported that electromagnetic
field decreased total protein content of Corn. However, the used SDS-PAGE to assay protein content. Touati et
al also reported increasing of catalase activity, in Radish cotyledons, induced by moderate static magnetic field
[36]. Peroxidase activity was found to be increased by magnetic fields in agriculture plants [27]. But here, the
results of the present study showed the highest peroxidase activity in control dry samples. However, it was
followed by 15min dry treated samples. Celik et al., reported that magnetic field increased activity of catalase
and superoxide dismutase in root samples of Glycine max [7].It is also indicated that activity of catalase and
superoxide dismutase, were increased by weak static electric and magnetic fields, in apoplastic and symplastic
areas of shallot leaves [6]. Abdollahi, F., V. Niknam, et al, infected Citrus aurantifolia by Candidatus
Phytoplasma aurantifoliae and then exposed samples to electromagnetic field.
The results showed protein content was increased in both non-infected and infected samples. However,
hydrogen peroxide was found to be decreased in non-infected and infected samples in comparison to control
samples [1]. Rammal et al reported decreasing of antioxidant potential of plant Lycopersicon esculentum by
assessment of DPPH radical scavenging of electromagnetic field treated samples [22]. However, in the present
study, it is shown that 30minuntes treatments of electromagnetic field brought about higher DPPH radical
scavenging, than control samples. Antioxidant properties such as DPPH, of Curcuma alismatifolia leaves, were
increased by gamma radiation [33]. Shivashankara et al, indicated that antioxidant parameters such as FRAP,
were not significantly changed ,by pretreatments of electric fields in fruits of mangoes [30]. Pulsed electric field
treatment of apple, did not increase some antioxidant parameters such as DPPH and FRAP in fruit juice [24].
However, based on the results of present study, both DPPH and FRAP were significantly increased by
electromagnetic field. Teh, S.-S., B. Niven, et al, also reported that pretreatment of Cannabis sativa by
Microwave and Pulsed Electric Field, enhanced DPPH˙ scavenging activity and ferric reducing/antioxidant
power (FRAP) [34].
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