Ameliorative effect of salicin against gamma irradiation induced

UK Journal of Pharmaceutical and Biosciences Vol. 3(2), 29-41, 2015 RESEARCH ARTICLE
Ameliorative Effect of Salicin Against Gamma Irradiation Induced Electrophoretic
Changes in Brain Tissue in Male Rats
Mohga Shafik Abdalla, Hayat Mohamed Sharada, Ibrahim Abulyazid, Monira Abdel-Latif Abdel-Karim,
Wael Mahmoud Kamel*
Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, Giza, Egypt-12622
Article Information
Received 10 Feburary 2015
Received in revised form 27 April 2015
Accepted 28 April 2015
Abstract
The aim of this study was to investigate the radioprotective effect of salicin against irradiation
effect on brain tissue of male rats. Lipid peroxidation level was measured as thiobarbituric acid
reactive substance in brain tissue. The polyacrylamide gel electrophoresis for native protein,
lipoprotein and zymogram were carried out in brain homogenate. As expected, salicin resisted
the irradiation effect and declined the MDA level in brain homogenate of all treated groups
(especially in the irradiated salicin post-treated group). Salicin minimized the qualitative
mutagenic effect of irradiation on the electrophoretic protein pattern in all irradiated salicin
treated groups and it showed the highest antagonistic effect against irradiation in irradiated
salicin post-treated group (SI = 0.57). It could not prevent the abnormalities occurred qualitatively
and quantitatively as a result of irradiation in lipoprotein pattern in all irradiated salicin treated
groups. In the electrophoretic esterase pattern, salicin prevented the qualitative effect of
irradiation in irradiated salicin post-treated group (SI = 1.00). Salicin minimized the qualitative
irradiation effect on the catalase pattern in the irradiated salicin pre-treated group (SI = 0.73).
While in the peroxidase pattern, salicin adminstration resisted the irradiation effect in the
irrradiated post-treated groups (SI = 0.67). The results suggested the radioprotective ability of
salicin against gamma irradiation effect on various electrophoretic patterns in brain tissue of male
rats.
Keywords:
Gamma irradiation,
Salicin,
Brain,
Protein electrophoresis,
Isozymes
*
Corresponding Author:
E-mail: wmkamel83@hotmail.com
Tel.: 00201126682686
1 Introduction
The use of this radiation as a source to enhance the mutation
frequency was recognized as early as 19301
.These rays act either
directly or by secondary reactions to produce biochemical lesions
that initiate series of physiological symptoms. They are known to
induce oxidative stress through the generation of reactive oxygen
species (ROS) resulting in imbalance of the prooxidant and
antioxidant activities, ultimately resulting in cell death2
.Studies
showed that radiation can affect a wide variety of tissues, particularly
those with greater levels of cellular turnover and divisions3
.
Irradiation causes similar damage at a cellular level but gamma rays
are more penetrating, causing diffuse damage throughout the body4
.
This type of rays also used for diagnostic purposes in nuclear
medicine in imaging techniques5
.
Irradiation causes damage to living tissue through a series of
molecular events. The formation of ROS as a result of interaction of
irradiation with cellular macromolecules is the cause of dysfunction
and death, in both normal as well as tumor cells exposed to
radiation6,7
.The energy exchange between the rays and the targeted
molecules leads to changes produced in deoxyribonucleic acid
(DNA), lipids, and proteins and then cell inactivation8,9
.
Gamma irradiation was found to interrupt energy supplies and
blocking all key enzymes, which may stop normal metabolism of the
exposed tissue10
. The radiation exposure whether occupational or
during radiotherapy leads to serious systemic damage to various
cellular and subcellular structures11
.
Irradiation causes induction of lipid peroxidation as evidenced by
increased malondialdhyde (MDA)12
. It causes damage of cells
UK Journal of Pharmaceutical and Biosciences
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ISSN: 2347-9442

Kamel et al. Ameliorative Effect of Salicin Against Gamma Irradiation
UK J Pharm & Biosci, 2015: 3(2); 30
directly by ionizing DNA and other cellular targets and indirectly by
effect through ROS13
. It produces oxygen-derived free radicals in
tissue environment: these include hydroxyl radicals (the most
damaging), superoxide anion radicals and other oxidants such as
hydrogen peroxide14
.
Major radiation damage is due to the aqueous free radicals
generated by the water radiolysis. These free radicals act as
molecular marauders and in turn damage DNA which is considered
to be the primary target15
.This gives rise to genomic instability
leading to mutagenesis, carcinogenesis and cell death16
.
The most important consequences of OS are lipid peroxidation,
protein oxidation and depletion of antioxidants17,18
. The latter authors
showed thatthe increase of MDA level is probably due to the
interaction of •OH resulting as a bi-product of water radiolysis with
the polyunsaturated fatty acids presents in the phospholipids portion
of cellular membranes. The excessive free radicals can damage
crucial macromolecules, including DNA, cell membranes and
enzymes, and can cause cell death. DNA damage includes
genotoxicity, chromosomal abnormalities, gene mutations and cell
death if the damage is beyond repair19,20
.
Catalase (CAT) and glutathione peroxidase (GPx) play an important
role in detoxification of hydrogen peroxide and ROS21
(Kula et al.,
2000). The irradiation caused significant increase in markers of the
oxidative stress as malondialdehyde (MDA) and alterations in activity
of antioxidant enzymes such as CAT and GPx in a dose-dependent
manner22,23
.
Salicin was discovered in 1831. It was isolated from the willow bark
and leaves. It is a prodrug and thus a precursor of salicylic
acid24
.Salicin is a phenolic glycoside. It exhibits analgesic effects,
and it is used for the treatment of rheumatic pain. Its occurrence in
willow (Salix) species is the major reason willow bark and its extracts
are popular products. High-Performance Liquid Chromatography
(HPLC) has been the standard method of analysis of quantifying
salicin in the different Salix species25
.
The present experiment was concerned with studying the ability of
salicin which represents the most abundant fraction of aqueous
willow extract to impress radiation-induced electrophoretic alterations
in brain tissue of male rats.
2 Materials and Methods
2.1 Salicin isolation
Fresh young leaves of the willow trees (Salix subserrata, Salix
safsaf) were collected from Orman garden, Giza, Egypt. This species
was well authenticated by qualified specialists in plant taxonomy.
Salicin was extracted and isolated from fresh leaves according to
method described by Mabry et al26
and purified according to method
suggested by Kur'yanov et al27
then identified qualitatively by
traditional and advanced chromatographic techniques.
2.2 Acute toxicity test
The lethal dose 50 (LD50) was evaluated on 6 groups of female
albino mice (8 animals / group) receiving progressively increasing
oral dose levels (500, 1000, 2000, 3000, 4000 and 5000 mg/kg body
weight) of aqueous solution of salicin solution. Mortality was
recorded 24 hrs post treatment. The LD50 was calculated according
to the equation suggested by Paget and Barnes28
.
2.3 Animals
Seven groups of male rats weighing between 150-200 gm per one
obtained from the animal house laboratory of the national research
centre. Ten rats in each group. All the animals were kept under
normal environmental and nutritional conditions. The animal groups
were divided into Control group: Rats were non-irradiated and non-
treated with salicin, Salicin treated group: Rats were non-irradiated
but treated with the safe dose of salicin which was about 150 mg /
Kg taking in the consideration weight of each rat, Irradiated group:
Rats were irradiated at the dose 7 Gy and non-treated with salicin,
Irradiated salicin pre-treated group: Rats were treated with salicin for
15 days followed by irradiation at the 15th
day, Irradiated salicin
prepost-treated group: Rats were treated with salicin for 15 days
followed by irradiation at the 15th
day then the treatment was
continued daily for another 15 days, Irradiated salicin simultaneous
treated group: Rats were irradiated and treated with salicin at the
same time of irradiation and continue daily for 15 days and Irradiated
salicin post-treated group: Rats were irradiated at the same gamma
dose then left without treatment for 15 days. At the 15th
day, the rats
were treated with salicin for another 15 days.
2.4 Irradiation
Whole body was gamma irradiated at Middle Eastern Regional
Radioisotope Centre for the Arab Countries, Dokki, Egypt. Using
Cobalt 60 (Co 60
) as a suitable gamma source. Rats were irradiated
at a single dose of 7 Gy delivered at the dose rate of 1.167 Rad /
Sec.
2.5 Biochemical Assay
Lipid peroxidation level was measured as thiobarbituric acid reactive
substance in brain homogenate according to method of Ohkawa et
al29
.
2.6 Electrophoretic protein pattern
Total protein was determined in brain homogenate according to
Bradford30
.The sample was mixed with the sample buffer. The
protein concentration in each well must be about 70 μg protein.
Proteins were separated through polyacrylamide gel electrophoresis

(PAGE) with different concentrations. Electrode and gel buffer and
polyacrylamide stock were prepared according to Laemmli31
. After
electrophoretic separation, the gel was gently removed from the
apparatus and put into a staining solution of coomasie brilliant blue
for native protein pattern32
and staining solution of sudan black B
(SBB) for lipoprotein pattern33
.
2.7 Electrophoretic isozyme
Native protein gel was stained for peroxidase pattern using certain
stain prepared according to the method suggested by Rescigno et
al34
. It was stained for catalase pattern according to the method
described by Siciliano and Shaw35
.For esterase pattern, the native
gel was stained according to the method suggested by Baker and
Manwell36
.
2.8 Data analysis
The polyacrylamide gel plate was photographed, scanned and then
analyzed using Phoretix 1D pro software (Version 12.3). The
similarity index (S.I.) compares patterns within, as well as, between
irradiated and non-irradtated samples. The similarity values were
converted into genetic distance (GD) according the method
suggested by Nei and Li37
.
2.9 Statistical analysis
All the grouped data were statistically evaluated with SPSS/16.00
software. The results were expressed as mean ± SE of studied
groups using the analysis of variance test (one-way ANOVA)
followed by student’s t-test34
. P values of less than 0.05 were
considered to indicate statistical significance. The means of
irradiated groups and the salicin treated groups were individually
compared with those of control group. The irradiated group was
compared with irradiated salicin treated groups
3 Results and Discussions
3.1 Lipid peroxidation
As compared to control, irradiation caused the severe increase in the
lipid peroxidation product (MDA) level in the brain tissue. Salicin
administration showed the ameliorative effect against irradiation by
reducing MDA level in all irradiated salicin treated rats. From the
data compiled in table 1, it was found that salicin showed the most
suitable antagonistic effect against irradiation on brain of irradiated
salicin post-treated group.
3.2 Electrophoretic protein pattern
Protein pattern in the control sample produced 9 bands with Rfs
ranged between 0.09 – 0.98 (Mwts 5.21 – 212.46 KDa and B %
values 0.20 – 25.09). There were no common bands but there were
2 characteristic bands appeared in irradiated salicin pre-treated
group with Rf 0.56 (Mwt 20.09 KDa and B % 21.13) and in irradiated
salicin simultaneous treated group with Rf 0.75 (Mwt 14.58 KDa and
B % 10.85).
As shown in table 2 and illustrated in fig. 1, irradiation caused
disappearance of 6 normal bands without appearance of abnormal
bands. The 3rd
and 5th
bands might be deviated to be appeared with
Rf 0.22 and 0.43 (Mwts 113.48 and 30.53 KDa and B % 17.28 and
80.91). It caused quantitative mutation represented by increasing B
% of the 9th
band (Rf 0.98, Mwt 5.02 KDa and B % 1.81). Salicin
administration resisted the qualitative mutagenic effect of irradiation
in the irradiated salicin simultaneous treated and post-treated
groups. It retained 4 normal bands with Rfs ranged between Rf 0.23 -
0.98 (Mwts 5.08 - 112.21 KDa and B % 0.20 - 15.81) in irradiated
salicin simultaneous treated group and with Rf 0.45 - 0.99 (Mwts 4.75
- 27.06 KDa and B % 0.59 - 56.41) in irradiated salicin post-treated
group. It was probable that the 5th
band was deviated to be appeared
with Rf 0.45 (Mwt 28.20 KDa and B % 37.70) in irradiated salicin
simultaneous treated group. It could not prevent the quantitative
mutation which was represented by increasing B % of the 3rd
and 7th
bands (Rfs 0.23 and 0.85, Mwts 112.21 and 10.65 KDa and B %
15.81 and 14.00) and by decreasing B % of the 6th
band (Rf 0.63,
Mwt 18.00 KDa and B% 11.99) in irradiated salicin simultaneous
treated group. Also, it could not prevent the quantitative mutation
which was represented by increasing B % of the 5th
, 6th
, 7th
and 9th
bands (Rfs 0.45, 0.65, 0.86 and 0.99 (Mwts 27.06, 17.68, 10.38 and
4.75 KDa and B % values 56.41, 0.59, 34.49 and 1.54) respectively.
It was found that the lowest SI value (SI = 0.17) was recorded with
irradiated group and the highest SI value (SI = 0.70) with salicin
treated group. Salicin improved the SI values in all irradiated salicin
treated groups, and it showed the highest antagonistic effect against
irradiation in irradiated salicin post-treated group (SI = 0.57). The
overall results showed that salicin prevented the irradiation effect on
number and arrangement of the bands in all irradiated salicin treated
groups.
3.3 Electrophoretic lipoprotein pattern
As revealed in table 3 and illustrated in fig. 2, lipoprotein pattern in
the control sample produced 3 bands with Rfs 0.22, 0.47 and 0.69 (B
% 67.28, 18.91 and 13.82). There were no common bands appeared
in all groups. Salicin alone caused severe qualitative alterations
represented by disappearance of the normal bands with appearance
of 3 abnormal bands with Rf 0.25, 0.33 and 0.72 (B % 48.27, 31.12
and 20.61).
Irradiation caused severe abnormalities represented disappearance
of all the normal bands without appearance of abnormal bands in the
irradiated, irradiated salicin simultaneous treated and post-treated
groups. Salicin adminstration could not prevent the irradiation effect

which was represented qualitatively by disappearance of 2 normal
bands and quantitatively by increasing B % of the normal band
appeared with Rf 0.22 and B % 100.00 in the irradiated salicin pre-
treated group and by disappearance of 2 normal bands with
appearance of one abnormal band with Rf 0.35 (B % 29.73) in the
irradiated salicin prepost-treated group. It was observed that all the
bands were not matched with all bands of the other groups in the
salicin treated, irrradiated, irradiated salicin simultaneous treated and
post-treated groups.
Table 1: Effect of irradiation, salicin and their combination in various treatment modes on the level of lipid peroxidation in spleen
tissue of male rats
Organ Control Sal. Irr.
Irradiated salicin treated groups
Pre-treated Simultaneous Prepost-treated Post-treated
Brain
(nmol/g)
162.02
± 2.28
137.84a
± 2.17
239.15a
± 6.55
232.90a
± 6.24
227.21a
± 4.78
228.10a
± 7.95
165.24a,b
± 2.60…
a : Different from control at P < 0.05, b : Different from the irradiated group at P < 0.05.
Note : Sal. ; salicin, Irr. : Irradiated
3.4 Electrophoretic esterase pattern
The electrophoretic esterase pattern in control of brain tissue
produced 3 types with Rfs 0.22, 0.48 and 0.64 (B % 36.85, 45.38 and
17.77). As shown in table 4 and illustrated in fig. 3, there were no
common bands in all groups. Salicin alone caused qualitative
mutation represented by appearance of one abnormal characteristic
band with Rf 0.11 (B % 21.93) and decreasing B % of the 2nd
normal
type (Rf 0.49 and B % 19.00). Irradiation caused disturbances
represented qualitatively by disappearance of 2 normal types of the
enzyme and quantitatively by increasing B % of the 1st
normal band
(Rf 0.21 and B % 100.00).
Salicin administration prevented the qualitative or quantitative
alterations occurred as a result of irradiation in the irradiated salicin
post-treated group. It could not prevent the irradiation effect which
was represented qualitatively by deviation of the 3rd
type to be
appeared with Rf 0.60 (B % 23.33) and quantitatively by increasing B
% of the 1st
band (Rf 0.21 and B % 51.38) and decreasing B % of the
2nd
type (Rf 0.47 and B % 25.29) in the irradiated salicin pre-treated
group, represented by disappearance of the 3rd
type with deviation of
the 1st
and 2nd
normal types of the enzyme to be appeared with Rfs
0.23 and 0.48 (B % 55.23 and 44.77) in the irradiated salicin
simultaneous treated group and represented by deviation of the 2nd
and 3rd
normal types of the enzyme to be appeared with Rfs 0.45 and

0.62 (B % 38.34 and 21.20) in the irradiated salicin prepost- treated
group.
Fig.1:Electrophoretic pattern showing effect of salicin against
the irradiation effect on protein pattern in brain tissue of male
rats
Fig. 2:Electrophoretic pattern showing effect of salicin against
the irradiation effect on lipoprotein pattern in brain tissue of
male rats
Table 3: Data of the electrophoretic lipoprotein pattern in brain tissue of control, irradiated and irradiated salicin treated groups in
male rats
Control Salicin Irradiated
Irradiated salicin treated
Rf. B. % Rf. B. % Rf. B. % Rf. B. % Rf. B. % Rf. B. % Rf. B. %
0.22 67.28 0.25 48.27 — — 0.22 100.00 — — 0.22 70.27 — —
0.47 18.91 0.33 31.12 — — — — — — 0.35 29.73 — —
0.69 13.82 0.72 20.61 — — — — — — — — — —
Rf.: Rate of Flow, B.%: Band Percent
Table 4: Data of the electrophoretic esterase pattern in brain tissue of control, irradiated and irradiated salicin treated groups in male
rats
0.22 36.85 0.11 21.93 0.21 100.00 0.21 51.38 0.23 55.23 0.21 40.46 0.22 40.27
0.48 45.38 0.22 40.13 — — 0.47 25.29 0.48 44.77 0.45 38.34 0.47 36.92
0.64 17.77 0.49 19.00 — — 0.60 23.33 — — 0.62 21.20 0.62 22.81
— — 0.64 18.94 — — — — — — — — — —

It was found that the highest SI value (SI = 1.00) was noticed in
irradiated salicin post-treated group. In the irradiated salicin
simultaneous treated group, it was observed that all the bands were
not matched with all bands of the other groups.
the irradiation effect on esterase pattern in brain tissue of male
rats
3.5 Electrophoretic catalase pattern
As shown in table 5 and illustrated in fig. 4, 5 types of catalase
enzyme were produced in the control sample with Rfs ranged
between 0.21 - 0.97 (B % 8.11 – 47.12). There were 2 common
bands appeared in all the groups with Rfs 0.45 and 0.97 (B % 8.47
and 15.17). Salicin alone caused alterations represented by
appearance of one abnormal band with Rf 0.18 (B % 23.47) with
decreasing the B % of the 1st
type (Rf 0.22 and B % 6.12) and
increasing the B % of the 2nd
type (Rf 0.44 and B % 25.89).
Irradiation caused qualitative alterations represented by
disappearance of the 1st
type of the enzyme with deviation of the 3rd
band to be appeared with Rf 0.78 (B % 15.01) and quantitative
mutation occurred by increasing B % of the 2nd
band (Rf 0.45 and B
% 64.42).
Salicin administration could not prevent the irradiation effect which
was represented by alteration occurred qualitatively by appearance
of one abnormal band with Rf 0.15 (B % 29.71) with deviation of the
3rd
type of the enzyme to be appeared with Rf 0.82 (B % 28.32) and
quantitatively by decreasing B % of the band appeared with Rf 0.22
(B % 9.44) in the irradiated salicin pre-treated group, by
disappearance of the 1st
and 4th
types of the enzyme with appearance
of one abnormal band with Rf 0.17 (B % 31.52) and deviation of the
3rd
type to be appeared with Rf 0.82 (B % 38.60) in the irradiated
salicin prepost-treated group and represented by disappearance of
the 1st
type with appearance of one abnormal band with Rf 0.12 (B %
20.99) deviation of the 3rd
type to be appeared with Rf 0.83 (B %
19.39) in the irradiated salicin simultaneous treated group. While in
the irradiated salicin post-treated group, salicin could not overcome
the mutation represented qualitatively by disappearance of 3 normal
types with appearance of one abnormal band with Rf 0.66 (B %
57.05) and quantitatively by increasing B % of the bnds appeared
with Rfs 0.45 and 0.96 (B % 20.65 and 22.30).
From the SI values, it was found that the lowest SI value (SI = 0.44)
was observed with irradiated salicin prepost-treated group and the
highest value (SI = 0.91) observed with salicin treated group. Salicin
treatment minimized the qualitative irradiation effect in the irradiated
salicin pre-treated group (SI = 0.73) more than the other irradiated
salicin treated groups.
3.6 Electrophoretic peroxidase pattern
As shown in table 6 and illustrated in fig. 5, it was found that 5 types
of peroxidase enzyme were produced in the control sample with Rfs
ranged between 0.28 - 0.72 (B % 0.46 - 41.66). There were no
common band appeared in all groups. Salicin alone caused no
obvious qualitative or quantitative alterations. Irradiation caused
severe disturbances represented qualitatively by disappearance of
the 2nd
, 3rd
and 5th
normal types of the enzyme and quantitatively by
increasing B % of the 1st
type (Rf 0.28 and B % 58.28). Salicin could
not remove the abnormalities which were represented qualitatively
by disappearance of the 1st
, 3rd
and 5th
types with increasing B % of
the 2nd
and 4th
normal types of the enzyme (Rfs 0.35 and 0.58 and B
% 34.34 and 65.66) in irradiated salicin pre-treated group and
represented qualitatively by disappearance of the 3rd
type of the
enzyme with appearance of one abnormal characteristic band with Rf
0.69 (B % 44.83) and quantitatively by decreasing the B % of the 1st
type (Rf 0.27 and B % 14.08) and increasing B % of the 2nd
and 4th
types of the enzyme (Rfs 0.38 and 0.69 and B % 25.82 and 44.83) in
irradiated salicin prepost-treated group. In the irradiated salicin post-
treated group, salicin could not prevent the mutagenic effect which
was represented qualitatively by disappearance of the 5th
type and
deviation of the 4th
type to be appeared with Rf 0.56 (B % 61.82) and
quantitatively by decreasing B % of the 1st
type appeared with Rf
0.27 and 14.47.
From the SI values, it was found that salicin minimized the irradiation
effect on the band number and arrangement in the irradiated salicin
prepost-treated groups (SI = 0.44) and post-treated groups (SI =
0.67). In the irrradiated pre-treated group, it was observed that all the
bands were not matched with all bands of the other groups. It
showed the highest antagonistic effect in the irradiated salicin post-
treated group.
4 Discussions

During results of the present study, the MDA level elevated
significantly as a result of radiation exposure in the brain tissue. This
was in accordance with the results obtained by Saada and Azab39
that showed that the MDA level increased due to production of ROS
associatedwith the increase in lipid peroxidation. ROS are known to
attack the highly unsaturated fatty acids of the cell membrane to
induce peroxidation reactions, which considereda key process in
many pathological events and is one ofthe reactions induced by
oxidative stress40
. The increasein intracellular ROS concentration
leads subsequently tooxidative stress41
and decrease in activity of
antioxidantenzymes with possible damage of cellular membranes42
.
The present study showed that irradiation increased the MDA level.
This was in agreement with Dixit et al 43
who showed that the doses
2, 6 and 10 Gy of irradiation enhanced the MDA level. This may be
due to reducing the antioxidant enzymes as superoxide dismutase,
catalase and glutathione-S-transferase.
It was demonstrated that brain belonged to the most biosensitive
organs to low doses of irradiation in rats44
. The oxidative stress
affects some brain regions more than others45
.
Irradiation induced lesions tend to occur more frequently in the
cerebral brain white matter. It caused necrosis in the cortex and
subcortical area in the brain46
. This because white matter tissue is
more affected than gray matter tissue47
. This means that the brain
represents one of the most important targets of irradiation. So, it was
selected to be under study during the current experiment.
The elevation of the levels of antioxidant enzymes in the irradiated
animals when compared with the control suggests that these
enzymes were up regulated to respond to the toxicity induced by the
radiation48
.
Proteins are the most complex compounds and at the same time the
most characteristic of living matter. They are present in all viable
cells; they are the compounds which, as nucleoproteins, are
essential for cell division and, as enzymes and hormones, control
many chemical reactions in the metabolism of cells. Thus, the
separation and characterization of the individual proteins facilitate
the study of the chemical nature and physiological function of each
protein49
.
Changes in the protein patterns of the tissues may reflect
specialization and adaptation in the organisms. It is worthy to note
that each protein is considered as reflect to the activity of specific
gene through the production of enzyme, which act as a catalyst to
produce the demanded protein; this type of produced protein is
responsible for a specific biological character50
. Proteins are major
targets for oxidative damage due to their abundance and rapid rates
of reaction with a wide range of radicals and excited state species51
.
It is worthy to note that each protein type has a biological role, due to
this role, the DNA secrets enzymes which act as catalysts to produce
the specific type of protein. Oxidative protein damage could also
affect the activity of DNA repair enzymes. Another possible
mutagenic effect of ROS involves their attack on lipids, to initiate lipid
peroxidation. The peroxides can decompose to a range of mutagenic
carbonyl products52
.
The proteins in brains of all rats were separated electrophoretically
and abundances were measured by using image analysis technique.
Data in the present study indicated that specific protein bands in
tissues of the irradiated rats differed (through disappearence in some
protein bands or appearance of new ones). Disappearance of some
protein bands in treated rats may be attributed to the effects of
irradiation, which inhibits the synthesis and expression process of
these deleted proteins (qualitative effect). In addition, even the band
remained after irradiation, it usually differs in the amount of protein,
and this may be explained by that irradiation could not inhibit the
synthesis of this protein type, but it may be affected only on the
quantitative level.
The representative electrophoretic profile of the proteins was carried
out in serum and different tissue homogenates. This profile showed
different types of mutations occurred as a result of radiation
exposure. As compared to control, some normal proteins were
represented by the fastest migrating band and had the highest
staining intensity. There were some proteins showed the
intermediate bands and the slowest migrating bands. On the other
hand, there was type of proteins represented as the biggest protein
band close to the well comb (origin). All these proteins differ from
each other in the mobility, molecular weight, optical density and band
intensity of each protein band.
In the present study, the similarity index between the control and all
the irradiated samples and between the irradiated samples
themselves recorded low values, indicating to apparent effect of the
irradiation and the differences in the protein pattern. It was stated by
many previous studies that the irradiation created a great genetic
distance between the control and the irradiated samples that may be
due to the activation of some genes. These genes produce different
types of proteins not produced in the control. These protein types
may lead to variation of the different biological processes.
The current study showed that there were different mutations was
detected by the appearance of new proteins or by the quantitative
decrease in abundance of normally occurring proteins. This was in
agreement with the results reported by Giometti et al 53
who reported
that the electrophoresis can be used to detect the mutations
reflected as quantitative changes in the protein expression.

Table 5: Data of the electrophoretic Catalase pattern in brain tissue of control, irradiated and irradiated salicin treated groups in male
rats
0.21 47.12 0.18 23.47 0.45 64.42 0.15 29.71 0.12 20.99 0.17 31.52 0.45 20.65
0.45 8.47 0.22 6.12 0.78 15.01 0.22 9.44 0.45 41.66 0.44 13.82 0.66 57.05
0.85 21.13 0.44 25.89 0.92 8.84 0.45 9.96 0.83 19.39 0.82 38.60 0.96 22.30
0.92 8.11 0.85 26.36 0.97 11.74 0.82 28.32 0.92 6.94 0.97 16.07 — —
0.97 15.17 0.93 7.04 — — 0.91 9.16 0.95 11.02 — — — —
— — 0.97 11.12 — — 0.97 13.41 — — — — — —
Table 6: Data of the electrophoretic peroxidase pattern in brain tissue of control, irradiated and irradiated salicin treated groups in
male rats
0.28 35.62 0.27 35.91 0.28 58.28 0.35 34.34 0.27 11.68 0.27 14.08 0.27 14.47
0.36 12.62 0.37 11.01 0.59 41.72 0.58 65.66 0.36 28.20 0.38 25.82 0.37 14.82
0.49 9.63 0.47 12.02 — — — — 0.57 60.12 0.58 15.28 0.48 8.89
0.59 0.46 0.60 0.63 — — — — — — 0.69 44.83 0.56 61.82
0.72 41.66 0.72 40.44 — — — — — — — — — —
the irradiation effect on catalase pattern in brain tissue of male
rats
the irradiation effect on peroxidase in brain tissue of male rats

During the current experiment, irradiation-dependent accumulation of
oxidatively modified proteins was studied in the brain tissues.
Alterations in the protein increased as a result of irradiation. This
may be related to an extensive modification of lysine and arginine
residues in histone molecules54
. The authors observed activation of
histone-specific proteases in the nuclei of γ-irradiated rats. The lack
of carbonyl accumulation in the nuclear proteins isolated from tissues
of γ-irradiated animals may be explained by the degradation of
oxidized histones by these proteases.
All lipoproteins carry all types of lipid, but in different proportions, so
that the density is directly proportional to the protein content and
inversely proportional to the lipid content55
. The lipoproteins were
more susceptible to oxidative modifications resulting in small
lipoproteins56
. The ROS can initiate one-electron oxidation or one-
electron reduction reactions on numerous biological systems. The
oxidative hypothesis classically admits the involvement of the
lipoproteins oxidation radiolytically57
. There was natural binding
between protein and lipoproteins in the rat tissues. These two tissues
known to be involved in the processing of the lipoproteins. The
lipoproteins-binding protein has previously been identified in adrenal
cortical plasma membranesandconcentration of the binding protein
was strongest in kidneys58
. So the alterations in the protein pattern
were associated with altering the lipoprotein pattern in these tissues.
The alterations in the lipoprotein pattern may refer to the
disturbances in the cholesteryl esterase required or cholesterol
hydrolysis. It was suggested that non-parenchymal liver cells
possess the enzymic equipment (cholesteryl esterase) to hydrolyze
very efficiently internalized cholesterol esters and this supported that
these cell types are an important site for lipoprotein catabolism59,60
.
Salicin administration showed the protective effect against the
irradiation. This may be due to its antioxidative effect against attack
of the free radicals. It prevented the alterations in the proteins and
hence the lipoproteins fractions in the brain tissues.
In the current experiment, polyacrylamide gel electrophoresis was
used for separation of different enzymes, which help in explanation
of different biological processes that occur inside the living
organisms due to the deleterious effect of irradiation and the
ameliorated effect of salicin.
According to the present data, the activities of CAT and GPx in
brains of irradiated rats were altered. These results strongly
suggested that irradiation has the capability to induce free radicals
and oxidative damage as evidenced by perturbations in various
antioxidant enzymes. Depletion of these enzymes activity could be
due to the direct effect on the enzymes by irradiation-induced ROS
generation, direct inhibition of the enzymes by irradiation61
.
The present work evaluated the enzymatic activity of the AO
enzymes enzymes as CAT and GPx and electrophoretic detection of
oxidative damage in proteins (carbonyl groups) in brains of rats. This
was in agreement with Ehrenbrink et al62
. The authors suggested
that the patterns of activity and accumulation of damages can be
sex-specific and related to the cycle of reproductive life of the rats.
Esterases with active thiol groups detected electrophoretically in
brain of different animal species. It exhibits wide distribution among
these species63
.The brain was selected to show the esterase activity
because it is rich in esterase enzyme due to its role in the
neurotransmission and communication of messages64
.
Electrophoresis plays a major role in identifying esterases. So, this
technique was selected to identify the esterase types in male and
female reproductive organs. The present study showed that
irradiation caused alterations in the electrophoretic esterase pattern
representing great differences in a number of zones of esterase
activity and in substrate specificity between all treated groups
compared to control.
During the current the experiment, irradiation caused alterations in
the electrophoretic esterase pattern. This may refer to effect of
irradiation on the protein pattern. As regards changes in
electrophoretic mobility demonstrated in the present study, it seemed
that free radicals affect the integrity of the polypeptide chain in the
protein molecule causing fragmentation of the polypeptide chain due
to sulfhydral-mediated cross linking of the labile amino acids as
claimed by Bedwell et al65
. The changes in the fractional activity of
different isoenzymes seemed to be correlated with changes in the
rate of protein expression secondary to DNA damage initiated by
free radicals66
.
During the current experiment, salicin administration showed the
highest ameliorative effect against irradiation in the irradiated salicin
treated rats. This may be due to trapping of these free radicals by
salicin, thus preventing DNA damage. Salicin and salicylic acid
belonged to the phenolic compounds which showed AO activity due
to their ability to scavenge free radicals67
.So, they are able to
overcome the disturbances in the esterase pattern in the tissues
selected to be under study.
Salicin belonged to the phenolic glycosides which are characterized
by their antioxidant activity in biological systems. The antioxidant
activity of the phenolic compounds refers to their ability to scavenge
free radicals67,68
. The authors suggested that the phenolic molecules
undergo redox reactions because phenolic hydroxyl groups readily
donate hydrogen to reducing agents. Ronca et al68
showed that
antioxidants suppressed hydroxyl radical production in the fenton
reaction, probably by chelating the iron required in the generation of
hydroxyl radicals. Results of the present study were in agreement

with that reported by Das et al69
who confirmed that elevation of MDA
concentration might be a consequence of decreased production of
antioxidants in the irradiated rats’ tissues. The antioxidant activities
of salicin fraction varied markedly. This may be due to the
differences in structures of phenolic compounds and primarily related
to their hydroxylation and methylation patterns70
.The total antioxidant
activity of the total aqueous extract of willow leaves was much more
than salicin alone. This may be due to the aqueous extract of willow
leaves contain other antioxidants such as tannins, flavenoids and
proanthocyanidins in addition to salicin which could play some role in
the increase of the total antioxidant activity71
. Salicin may induce
elevation in activities of the antioxidants as glutathione peroxidase in
the tissues homogenates which were selected to be under study.
The antioxidants have a major role in the antioxidants defense
mechanisms against irradiation injury72
.
So, the maintenance of normal electrophoretic protein and
zymogram levels after the treatment with salicin may be due to
trapping of these free radicals by this fraction, thus preventing DNA
damage. Salicin was able to overcome the disturbances in the
protein pattern in the tissues selected to be under study.
5 Conclusions
The study concluded that salicin showed radioprotective effect
against the gamma irradiation effect on various electrophoretic
patterns in brain tissue of male rats.
6 Competing interest
The study aimed to suggest new compounds obtained from the
nature and act as radioprotector to minimize or resist the irradiation
effect.
7 Author’s contributions
MSA and MALAK carried out literature review and draft the
manuscript. HMS participated in collection of data and arranged in
tabular form. IA and WMK carried out the experimental work. All
authors read and approved the final manuscript.
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