ELECTROPHORETIC STUDY OF THE ANTAGONISTIC EFFECT OF ...

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Kamel et al. World Journal of Pharmacy and Pharmaceutical Sciences

ELECTROPHORETIC STUDY OF THE ANTAGONISTIC EFFECT OF

SALICIN ISOLATED FROM EGYPTIAN WILLOW LEAVES (SALIX

SUBSERRATA) AGAINST THE EFFECT OF GAMMA IRRADIATION

IN MALE RATS

1Hayat M. Sharada,

2Mohga S. Abdalla,

3Ibrahim A. Ibrahim,

4Monira A. Abd El

Kader and 5*Wael M. Kamel

1,2Professor of Biochemistry, Faculty of Science, Helwan University.

3Professor of Molecular Biology, Biological Application Department, Atomic Energy

Authority. 4Assistant Professor of Clinical Biochemistry, Biochemistry Department, Division of Genetic

Engineering and Biotechnology, National Research Centre, 33 Bohouth St., Dokki, Giza,

Egypt, affiliation ID: 60014618. 5Researcher Assistant of Biochemistry, Biochemistry Department, Division of Genetic

Engineering and Biotechnology, National Research Centre, 33 Bohouth St.,

Dokki, Giza, Egypt, affiliation ID: 60014618.

OBJECTIVE

The study aimed to study efficiency of salicin to ameliorate irradiation

effect on various electrophoretic protein and zymogram patterns in

rats. Materials and Methods: The polyacrylamide gel electrophoresis

for native protein, lipoprotein and zymogram (esterase, catalase and

peroxidase) were carried out in serum samples of all groups. Results:

Irradiation caused various abnormalities in all electrophoretic patterns

(protein, lipoprotein and zymogram). It caused qualitative alterations

represented by disappearance of some or all normal bands with

appearance of abnormal bands and /or deviation of normal bands to be

appeared with another data (Rfs, Mwts and B % values). It caused

quantitative alterations represented by changing B % of the bands

appeared with normal Rf and Mwts. For electrophoretic protein

pattern, salicin administration reduced the irradiation effect on serum

sample of the irradiated salicin post-treated group (SI = 0.70) and for

lipoprotein pattern, salicin decreased the irradiation effect in irradiated

salicin pre-treated group (SI = 0.80). While in the electrophoretic zymogram, salicin

administration showed no antagonestic effect against the qualitative and quantitative

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Article Received on

18 March 2015,

Revised on 09 April 2015,

Accepted on 30 April 2015

*Correspondence for

Author

Wael Mahmoud Kamel

Researcher Assistant of

Biochemistry,

Biochemistry Department,

Division of Genetic

Engineering and

Biotechnology, National

Research Centre, 33

Bohouth st., Dokki, Giza,

Egypt, affiliation ID:

60014618.


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mutagenic effect of irradiation in all irradiated salicin treated groups. Conclusion: The

results showed that salicin prevented the irradiation effect on electrophoretic protein pattern

in the irradiated salicin post-treated group and lipoprotein pattern of irradiated salicin pre-

treated group. While in the electrophoretic zymogram, salicin could not prevent the

irradiation effect in all irradiated salicin treated groups.

KEYWORDS: Gamma rays, Rats, Protein pattern, Lipoprotein, Enzyme electrophoresis.

INTRODUCTION

All types of rays cause similar damage at a cellular level but gamma rays are more

penetrating, causing diffuse damage throughout the body (Bock, 2008). This type of rays also

used for diagnostic purposes in nuclear medicine in imaging techniques (Dwyer and David,

2012). Gamma irradiation was found to interrupt energy supplies and blocking all key

enzymes which may stop normal metabolism (Thornburn, 1972).

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 primary target (Arora et al., 2005). The effects of irradiation are cumulative and give

rise to genomic instability leading to mutagenesis, carcinogenesis, cell death, genetic damage

and numerous forms of body tissue pathology (Elshazly et al., 2012 ; Rubner et al., 2012).

At the cellular level, irradiation can induce oxidative stress or excessive production of ROS

and damage biologically important macromolecules such as DNA, proteins, lipids and

carbohydrates in the various organs and have been implicated in etiology of many diseases

(Halliwell and Gutteridge, 1989 ; Dixit et al., 2012). Oxidative stress was postulated as one

of the mechanisms of radiation toxicity. It leads to the development of a complex, dose-

dependent series of changes including changes in the structure and function of cellular

components and organs damage (Finkel and Holbrook, 2000). The most important

consequences of oxidative stress are lipid peroxidation, protein oxidation and depletion of

antioxidants (Spitz et al., 2004 ; Fedorova et al., 2010). The latter authors showed that the

increase of lipid peroxidation product level is probably due to the interaction of •OH resulting

as a bi-product of water radiolysis with the polyunsaturated fatty acids present 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.


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DNA damage includes genotoxicity, chromosomal abnormalities, gene mutations and cell

death if the damage is beyond repair (Seyed, 2010 ; Zhang et al., 2011).

It was well known that irradiation induced oxidative stress and generation of extra reactive

oxygen species and free radicals which attack sensitive enzymes, constitutive proteins, DNA

and membrane lipids (Mikkelsen and Wardman, 2003 ; Tominaga et al., 2004 ; Blatter and

Herrlich, 2004). The radiationinduced alteration of the protein structure was observed by

measuring the changes in the molecular properties of the proteins (Cho and Song, 2000 ;

Moon and Song, 2001). Whole-body irradiation showed significant increase in protein

carbonyls by 73% (Smutná et al., 2013). Altered protein molecules can act as traps for

chemical energy released by free radicals and initiate further chain reactions, thus enhancing

the damage as observed with lipid peroxides. Advanced oxidation protein products are

reliable markers of the degree of protein damage in oxidative stress (Witko-Sarsat et al.,

1999).

Among the antioxidant enzymes, SOD, CAT, GPx and GST are the first line of defense

against oxidative injury. These enzymes normally act as a team (Lee et al., 2007). SOD is the

primary step of the defense mechanism in the antioxidant system against OS by catalyzing

the dismutation of 2 superoxide radicals (O2 _) into molecular oxygen (O2) and hydrogen

peroxide (H2O2) (Gupta, 2006). H2O2 is neutralized by the combined action of CAT and GPx

in all vertebrates (Salvi et al., 2007). These enzymes act in coordination and the cells may be

pushed to OS state if any change occurs in the levels of enzymes (Attia et al., 2012).

Antioxidants eliminate the free radicals and neutralize ROS ions before they can do their

damage. However, much remains unknown about mechanisms of radio-protection.

Development of protective agents presented new solutions for recovery of undesired tissue

damage induced by irradiation (Kim et al., 2003 ; Elshazly et al., 2012).

Radioprotective agents are compounds that are administered before exposure to ionizing

radiation to reduce its damaging effects, including radiation-induced lethality (Stone et al.,

2004). The discovery of radioprotectors for the first time seemed to be very promising and

has attracted the interest of a number of radiobiologists. Although synthetic radioprotectors

such as the aminothiols have yielded the highest protective factors; typically they are more

toxic than naturally occurring protectors (Weiss and Landauer, 2003). Thereafter different


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plant extracts were tested against radiation effects and showed potential radioprotective

activities in mammals (Landauer et al., 2001 ; Jagetia and Baliga, 2004).

The willow trees are sources of salicin, which has analgesic as well as anti-inflammatory

properties. Salicylic acid, released from salicin in the body, provides anti-inflammatory and

pain-relieving actions (Pilotto et al., 2004) including the pain associated with knee and/or hip

osteoarthritis (Bigler et al., 2001) and back pain (Macarthur et al., 2005). It has similar

inhibitory effect on COX-2 as aspirin, but unlike aspirin it does not function as an

anticoagulant (Hawkey, 2004).

Salicin is a natural product extracted from several species of Salix (willow) and Populus

(poplar), and was also found in Gaultheria procumbens (wintergreen) and in Betula lenta

(sweet birch) (Jourdier, 1999). The pharmacokinetic and pharmacological properties of

salicin have made it an ideal antipyretic prodrug (Akao et al., 2002). It is considered as

natural aspirin. It is very possible to be digested without side effects in the stomach and

kidneys, while acetylsalicylic acid is known to upset the stomach and in some cases damage

kidneys. Scientists believe that this is because salicin is converted to acetylsalicylic acid after

the stomach has absorbed it (Vane et al., 1990).

Salicin hydrolyzed in the gastrointestinal tract to give D-glucose and salicyl alcohol. Upon

absorption, salicyl alcohol is oxidized into salicylic acid and other salicylates compounds

such as saligenin, salicyluric acid, salicyl glucuronides, and gentisinic acid which all are

eliminated through the kidney (Chrubasik and Shvartzman, 1999; Chrubasik and Eisenberg,

2004). Salicin belonged to the phenolic compounds which are believed to work

synergistically to promote healthy conditions through a variety of different mechanisms, such

as enhancing antioxidant activity, impacting cellular processes associated with apoptosis,

platelet aggregation, blood vessel dilation, and enzyme activities associated with carcinogen

activation and detoxification (Singh et al., 2009 ; Nzaramba et al., 2009).

MATERIALS AND METHODS

Fresh young leaves of the willow trees (Salix subserrata, Salix safsaf) were collected from

Orman garden, Giza, Egypt. Salicin was isolated according to method describe by Mabry et

al. (1970) and purified according to method suggested by Partridge (1949) and improved by

Kur'yanov et al., 1991) then identified qualitatively by the advanced chromatographic

techniques.


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Acute toxicity test

The lethal dose 50 (LD50) was evaluated on 6 groups of mice female albino mice of 21 – 25

gm body weight (8 animals / group) receiving progressively increasing oral dose levels of

salicin solution which was administrated orally in different doses to find out the range of

doses which cause 0 and 100% mortality of animals. The LD50 was calculated according to

the equation suggested by Paget and Barnes (1974).

Animals

Seven groups of male rats weighing between 150-200 gm per one obtained from the animal

house laboratory of national research centre. Ten rats in each group. All the animals were

kept under normal environmental and nutritional conditions. The animal groups were divided

as the following : rats were non-irradiated and non-treated with salicin representing control

group, rats were non-irradiated but treated with the safe dose of salicin (was about 150 mg /

Kg) taking in the consideration weight of each rat representing salicin treated group, rats

were irradiated at the dose 7 Gy and non-treated with salicin representing irradiated group,

rats were treated with salicin for 15 days followed by irradiation at the 15th

day representing

irradiated salicin pre-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

representing irradiated salicin prepost-treated group, rats were irradiated and treated with

salicin at the same time of irradiation and continue daily for 15 days representing irradiated

salicin simultaneous treated group and 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 representing irradiated salicin post-treated group.

IRRADIATION

Irradiation was carried out at Middle Eastern Regional Radioisotope Centre for the Arab

Countries, Dokki, Egypt. Rats were irradiated using Cobalt 60 (Co60) as a suitable gamma

source at single dose of 7 Gy delivered at the dose rate of 1.167 Rad / Sec.

Electrophoretic protein pattern

Total protein was determined in serum samples according to Bradford, (1976). 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 Laemmli, (1970). After electrophoretic separation, the gel was gently removed


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from the apparatus and put into a staining solution of coomasie brilliant blue for native

protein pattern (Hames, 1990) and staining solution of sudan black B (SBB) for lipoprotein

pattern (Chippendale and Beck, 1966).

Isozyme

Native protein gel was stained for esterase pattern, the native gel was stained according to the

method suggested by Baker and Manwell (1977). It was stained for catalase pattern according

to the method described by Siciliano and Shaw (1976). For peroxidase pattern using certain

stain prepared according to the method suggested by Rescigno et al., (1997).

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 Li (1979).

RESULTS

Electrophoretic protein pattern

Protein pattern in control sample produced 11 bands with Rfs ranged between 0.06 – 0.73

(Mwts 15.87 – 267.54 KDa and B % values 0.25 – 32.27) in control sample. As shown in

table 1 and illustrated in fig 1, there were 3 common bands in all groups with Rfs 0.49, 0.54

and 0.73 (Mwts 28.13, 23.34 and 15.87 KDa and B % 4.14, 11.52 and 32.27). There were 3

characteristic bands appeared individually in control group with Rf 0.30 (Mwt 85.43 KDa

and B % 0.25), in irradiated group with Rf 0.87 (Mwt 11.66 KDa and B % 17.50) and in

irradiated salicin simultaneous treated group with Rf 0.68 (Mwt 16.88 and B % 2.53).

Irradiation caused qualitative alterations represented by disappearance of 2 normal bands

with appearance of 3 abnormal bands with Rfs 0.23, 0.59 and 0.87 (Mwts 137.87, 19.70 and

11.66 KDa and B % 23.14, 7.00 and 17.50) respectively and quantitative mutation

represented by decreasing B % of the 1st, 3rd, 5th,9th and 11th bands appeared with Rfs 0.06,

0.27, 0.33, 0.54 and 0.75 (Mwts 267.54, 104.76, 64.78, 23.47 and 15.48 and B % values 3.93,

6.23, 0.44, 7.82 and 1.10) and by increasing B % of the 7th band appeared with Rf 0.44 (Mwt

33.82 and B % 19.62).


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From the similarity indix values, salicin could not prevent the disturbances in number and

arrangement of the bands in all irradiated salicin treated groups. It was found that number of

bands disappeared was lower in all irradiated treated groups than irradiated group. It was

found that the lowest SI value (SI = 0.43) was recorded with irradiated salicin prepost-treated

group and the highest value (SI = 0.91) was observed with salicin treated group. Salicin could

not overcome the qualitative alterations in protein pattern in all irradiated salicin treated

groups. It minimized the irradiation effect in the irradiated salicin post-treated group

(SI=0.70).

Fig. 1: Electrophoretic pattern showing effect of salicin against the irradiation effect on

protein pattern in serum sample of male rats.


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Table 1: Data of the electrophoretic protein pattern in serum sample of control, irradiated and irradiated salicin treated groups at different therapeutic modes in male rats.

Rf. : Rate of Flow, Mwt. : Molecular Weight, B. % : Band Percent.

Note : Arrangement of the bands at each lane is not correlated with the other bands in the other lanes.

Irradiated salicin treated

Control

Salicin

Irradiated

Pre-treated

Simultaneous Prepost-treated Post-treated

Rf. Mwt B. % Rf. Mwt B. % Rf. Mwt B. % Rf. Mwt B. % Rf. Mwt B. % Rf. Mwt B. % Rf. Mwt B. %

0.06 267.54 10.78 0.06 269.93 2.88 0.06 267.54 3.93 0.04 279.51 2.09 0.05 273.52 2.44 0.05 273.52 2.49 0.05 272.33 2.33

0.14 202.88 0.37 0.14 204.08 12.34 0.23 137.87 23.14 0.23 136.68 20.33 0.15 195.70 9.88 0.17 183.60 11.60 0.14 207.67 9.22

0.26 114.15 15.21 0.19 163.07 7.70 0.27 104.76 6.23 0.28 98.95 5.07 0.23 137.87 11.80 0.21 147.44 6.55 0.18 177.56 3.70

0.30 85.43 0.25 0.27 104.76 5.32 0.33 64.78 0.44 0.33 64.78 0.55 0.28 100.10 5.61 0.25 122.41 4.30 0.24 129.53 8.35

0.33 62.29 3.42 0.33 63.94 5.46 0.44 33.82 19.62 0.49 27.66 21.92 0.35 57.06 12.45 0.34 59.20 3.58 0.27 104.76 5.73

0.38 44.07 9.42 0.38 44.07 4.51 0.49 27.50 5.31 0.54 23.47 9.38 0.46 31.38 2.99 0.46 31.38 15.70 0.34 61.49 0.31

0.44 33.82 6.61 0.43 34.74 6.10 0.54 23.47 7.82 0.62 18.27 9.92 0.49 27.81 5.00 0.50 27.35 4.71 0.49 27.50 22.13

0.49 28.13 4.14 0.50 27.35 5.63 0.59 19.70 7.00 0.75 15.64 30.75 0.54 23.34 3.41 0.54 22.83 5.43 0.55 22.46 18.66

0.54 23.34 11.52 0.54 22.83 11.17 0.65 17.58 7.92 — — — 0.57 20.79 6.76 0.60 19.39 6.84 0.76 15.35 29.58

0.65 17.58 6.03 0.65 17.58 6.74 0.75 15.48 1.10 — — — 0.63 18.11 4.37 0.66 17.22 5.92 — — —

0.73 15.87 32.27 0.75 15.52 32.17 0.87 11.66 17.50 — — — 0.68 16.88 2.53 0.75 15.61 32.88 — — —

— — — — — — — — — — — — 0.74 15.75 32.77 0.75 15.61 0.00 — — —


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Electrophoretic lipoprotein pattern

Lipoprotein pattern in control sample produced 2 bands with Rfs 0.14 and 0.70 (B % 88.31

and 11.69). There was one common band appeared in all groups with Rf 0.70 and B % 11.69

(Table 2 and illustrated in fig. 2). Salicin alone caused alterations represented qualitatively by

disappearanc of the 1st band with appearance of 2 abnormal bands with Rfs 0.10 and 0.16

and B % 18.84 and 20.78 and quantitatively by increasing B % of the 2nd

band (Rf 0.72 and B

% 60.38). Irradiation caused qualitative alterations represented by deviation the 1st band to

be appeared with Rf 0.13 (B % 85.50). Salicin administration could not prevent the

irradiation effect which was represented by deviation the 1st band to be appeared with Rf

0.16 (B % 86.52) in irradiated salicin simultaneous treated group and with Rf 0.11 (B %

88.67) in the irradiated salicin post-treated group. Salicin administration could not prevent the

irradiation effect which was represented qualitatively by appearance of only one abnormal

band with Rf 0.57 (B % 60.90) and quantitatively by increasing B % of the 1st normal band

in the irradiated salicin pre-treated group and represented in the irradiated salicin prepost-

treated groupby appearance of 2 abnormal bands with Rfs 0.14 and 0.31 (B % 14.81 and

51.15).

It was found that the lowest SI values (SI = 0.40) was viewed with salicin treated group and

the highest SI values (SI = 0.80) was observed with irradiated salicin pre-treated group. As

compared to SI value of the irradiated group (SI = 0.50), salicin showed the most antagonistic

effect against irradiation in the irradiated salicin pre-treated group.

Fig. 2: Electrophoretic pattern showing effect of salicin against the irradiation effect

on lipoprotein pattern in serum sample of male rats.


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Table 2: Data of the electrophoretic lipoprotein pattern in serum sample of control, irradiated and irradiated salicin treated groups at

different therapeutic modes in male rats.


Control Salicin Irradiated

Pre-treated Simultaneous Prepost-treated Post-treated

Rf. B. % Rf. B. % Rf. B. % Rf. B. % Rf. B. % Rf. B. % Rf. B. %

0.14 88.31 0.10 18.84 0.13 85.50 0.15 32.59 0.16 86.52 0.08 24.79 0.11 88.67

0.70 11.69 0.16 20.78 0.72 14.50 0.57 60.90 0.73 13.48 0.14 14.81 0.72 11.34

— — 0.72 60.38 — — 0.72 6.51 — — 0.31 51.15 — —

— — — — — — — — — — 0.72 9.25 — —

Rf.: Rate of Flow, B. % : Band Percent.

Note: Arrangement of the bands at each lane is not correlated with the other bands in the other lane


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Electrophoretic esterase pattern

As revealed in table 3 and illustrated in fig. 3, 6 types of esterase enzyme were produced with

Rfs ranged between 0.10 - 0.79 (B % 8.42 - 23.76). There was one common band appeared in

all the groups with Rfs 0.56 (B % 22.94). Salicin alone caused qualitative mutation

represented by deviation of the 2nd and 3rd type of the enzyme to be appeared with Rfs 0.18

and 0.27 (B % 6.52 and 6.44) and quantitative mutation represented by increasing B % of the

4th type (Rf 0.56 and B % 47.22).

Irradiation caused qualitative mutation represented by disappearance of 2nd and 6th types

with appearance of one abnormal band with Rf 0.06 (B % 13.42) and also quantitative

mutation represented by decreasing B % of the 1st and 3rd types (Rfs 0.10 and 0.28 and B %

9.72 and 9.42) and increasing B % of the 4th type (Rf 0.58 and B % 55.15). Salicin could not

resist the qualitative and quantitative alterations occurred as a result of irradiation in all

irradiated salicin treated groups.

The SI values showed that the SI values (SI = 0.73) were equal in the irradiated and irradiated

salicin pre-treated groups. As compared to SI of the irradiated group (SI = 0.73), salicin

administration showed no antagonistic effect against irradiation in all irradiated salicin

treated groups.


esterase pattern in serum sample of male rats.


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Table 3: Data of the electrophoretic esterase pattern in serum sample of control, irradiated and irradiated salicin treated groups in male

rats.

Rf. : Rate of Flow, B. % : Band Percent.





0.10 20.09 0.09 19.69 0.06 13.42 0.05 9.58 0.56 77.48 0.10 26.90 0.05 10.21

0.20 8.42 0.18 6.52 0.10 9.72 0.09 5.98 0.72 10.24 0.58 50.98 0.10 15.12

0.29 23.76 0.27 6.44 0.28 9.42 0.58 58.98 0.80 12.28 0.71 9.74 0.59 51.99

0.56 22.94 0.56 47.22 0.58 55.15 0.70 11.36 — — 0.79 12.39 0.71 8.95

0.69 10.08 0.69 7.91 0.69 12.29 0.80 14.10 — — — — 0.80 13.73

0.79 14.72 0.80 12.22 — — — — — — — — — —


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Electrophoretic catalase pattern

The electrophoretic catalase pattern in control sample produced 2 types with Rfs 0.43 and

0.54 (B % 81.60 and 18.40). There were no common bands (Data recorded in table 4 and

illustrated in fig. 4). Salicin alone caused no quantitative mutation but it caused qualitative

alteration represented by appearance of one abnormal band with Rf 0.12 (B % 24.29).

Irradiation caused alterations in the catalase pattern represented qualitatively by

disappearance of the 1st normal type with appearance of 2 abnormal bands with Rfs 0.13 and

0.26 (B % 25.91 and 22.32) and deviation in the lase type to be appeared with Rf 0.51 (B %

51.77). Salicin administration could not prevent the irradiation effect which was represented

by disappearance of the 1st type of the enzyme with appearance of one abnormal band with

Rf 0.28 (B % 65.28) in the irradiated salicin pre-treated group, Rf 0.28 (B % 66.62) in

irradiated salicin simultaneous treated group, Rf 0.28 (B % 49.46) in irradiated salicin

prepost- treated group and Rf 0.26 (B % 59.73) in irradiated salicin post- treated group.

In the irradiated and irradiated salicin pre-treated groups, it was observed that all the bands

were not matched with all bands of the other groups. In the other irradiated salicin treated

groups showed the same SI value (SI = 0.50). There was complete similarity between these

groups.

Fig. 4: Electrophoretic pattern showing effect of salicin against the irradiation

effect on catalase pattern in serum sample of male rats.


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Table 4: Data of the electrophoretic Catalase pattern in serum sample of control, irradiated and irradiated salicin treated groups in

male rats.







0.43 81.60 0.12 24.29 0.13 25.91 0.28 65.28 0.28 66.62 0.28 49.46 0.26 59.73

0.54 18.40 0.44 55.37 0.26 22.32 0.51 34.72 0.52 33.38 0.53 50.54 0.52 40.27

— — 0.54 20.34 0.51 51.77 — — — — — — — —


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Electrophoretic peroxidase pattern

As shown in table 5 and illustrated in fig. 5, the electrophoretic peroxidase pattern in control

sample produced 3 types of enzyme with Rfs 0.40, 0.75 and 0.89 (B % 65.10, 21.61 and

13.29) respectively. There were no common bands appeared in all groups. The 1st type was

considered as common band in all groups except irradiated salicin simultaneous treated and

pre-treated groups. Salicin alone caused no quantitative mutation but it caused qualitative

lteration represented by deviation of the 3rd type to be appeared with Rf 0.89 (B % 13.29).

Irradiation caused severe alterations represented qualitatively by disappearance of the 2nd

type with deviation of the 3rd type to be appeared with Rf 0.92 (B % 13.43) and

quantitatively by increasing B % of the 1st type (Rf 0.40 and B % 86.57) and decreasing the

3rdtype (Rf 0.92 and B % 13.43). Salicin could not prevent the irradiation effect which was

represented qualitatively by disappearance of the 2nd type with appearance of one abnormal

band with Rf 0.49 (B % 71.93) with deviation of the 3rd type to be appeared with Rf 0.92 (B

% 12.88) and quantitatively by decreasing B % of the 1st type (Rf 0.40 and B % 15.19) in the

irradiated salicin pre-treated group, by disappearance of the 1st and 2nd types of the enzyme

with deviation of the 3rd type to be appeared with Rf 0.92 (B % 100.00) in the irradiated

salicin prepost-treated group, by disappearance of the 1st and 2nd types with appearance of

one abnormal band with Rf 0.33 (B % 86.95) with deviation of the 3rd type to be appeared

with Rf 0.91 (B % 13.05) in the irradiated salicin simultaneous treated group and represented

qualitatively by disappearance of the 2nd type with deviation of the 3rd type to be appeared

with Rf 0.93 (B % 96.87) and quantitatively by decreasing B % of the 1st type (Rf 0.39 and B

% 3.13) and increasing the 3rd type (Rf 0.93 and B % 96.87) in the irradiated salicin post-

treated groups.

The SI values (SI = 0.40) were equal in the irradiated and irradiated salicin post-treated

groups. There was complete similarity between these groups to each other and difference

from the control sample. In the irradiated salicin simultaneous treated and prepost-treated

groups, it was observed that all the bands were not matched with all bands of the other

groups. Salicin treatment could not prevent the irradiation effect on number and arrangement

of the bands in all irradiated salicin treated groups when compared to SI value of the

irradiated group (SI =0.40).


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peroxidase in serum sample of male rats.


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Table 5: Data of the electrophoretic peroxidase pattern in serum sample of control, irradiated and irradiated salicin treated groups in

male rats.





0.40 65.10 0.40 62.07 0.40 86.57 0.40 15.19 0.33 86.95 0.92 100.00 0.39 3.13

0.75 21.61 0.75 23.89 0.92 13.43 0.49 71.93 0.91 13.05 — — 0.93 96.87

0.89 13.29 0.91 14.04 — — 0.92 12.88 — — — — — —




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DISCUSSION

Salicin belonged to the phenolic glycosides which are characterized by their antioxidant

activity in biological systems. The antioxidant activity activity of the phenolic compounds

refers to their ability to scavenge free radicals (Madrigal-Carballo et al., 2009). The authors

suggested that the phenolic molecules undergo redox reactions because phenolic hydroxyl

groups readily donate hydrogen to reducing agents. The phenolic compounds act as reducing

agents (either by donating hydrogen atom or quenching the singlet oxygen), which explains

their antioxidant activities (Rice-Evans et al., 1996).

It was well known that irradiation was associated with many abnormal alterations occurred at

the molecular level including protein and lipoprotein pattern in addition to activity of some

enzymes as catalases, peroxidases and esterases. In 2011, Alabarse et al. reported that

oxidative stress was assessed by detecting the abnormal proteins, activity of antioxidant

enzymes as CAT and GPx in serum samples. They added that the activities of free radical

scavenging enzymes, including CAT and GPx were changed after irradiation.

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

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 proteins are responsible for a specific biological process, so due to the difference in

protein bands between all the treated samples, the biological processes may also be differed.

The separation and characterization of the individual proteins facilitate study of the chemical

nature and physiological function of each protein (Cheeseman, 1993).


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The current experiment showed that irradiation decreased the ordered structure of proteins

associated with increasing the irradiation dose. This was in agreement with Moon and Song,

(2001) who suggested that radiation caused initial fragmentation of polypeptide chains and,

as result, subsequent aggregation and degeneration of proteins by scavenging ROS produced

by irradiation. The difference in the protein fractions separated electrophoretically after

radiation exposure. The present study was in agreement with Pleshakova et al., (1998) who

reported that irradiation caused a rise of protein carbonyl only in the cytoplasm and

mitochondria and this was followed by activation of histone – specific proteases in nuclei of

the irradiated rats. The lack of carbonyl accumulation in the nuclear proteins in tissues of the

irradiated animals may be explained by the degradation of oxidized histones by these

proteases.

The present results showed that irradiation caused abnormalities in the electrophoretic protein

pattern. This was in accordance with many previous studies that showed that irradiation

caused irreversible changes at the molecular level by breakage of the covalent bonds of the

polypeptide chains. The exposure of proteins to oxygen radicals resulted in both non-random

and random fragmentations (Kempner, 1993). The protein fragmentation is affected by the

local conformation of an amino acid in the protein, its accessibility to the water radiolysis

products, and the primary amino acid sequence (Filali-Mouhim et al., 1997). It was reported

that irradiation caused aggregation and cross-linking of proteins. Covalent cross linkages are

formed between free amino acids and proteins, and between peptides and proteins in solution

after irradiation (Garrison, 1987 ; Filali-Mouhim et al., 1997). 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. (1987) who reported that the electrophoresis can

be used to detect the mutations reflected as quantitative changes in the protein expression.

Lipids are essential structural components of the cell membrane. They provide a rich source

of metabolic energy for periods of sustained energy demand (Chino et al. 1976). Most lipids

circulate through the bloodstream as lipoproteins. Lipoproteins are lipid–protein complexes

that contain large insoluble glycerides and cholesterol with a superficial coating of

phospholipids and proteins synthesized in the liver (Havel and Kane, 1995).

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 content


1595


(Bass et al., 1993). The lipoproteins were more susceptible to oxidative modifications

resulting in small lipoproteins (Tsumura et al., 2001).

There was natural binding between protein and lipoproteins. 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 membranes and concentration of the

binding protein was strongest in kidneys (Fidge, 1986). 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 catabolism (Theo et al., 1980 ; Satoh, 2005).

The present results showed that irradiation caused alterations in the electrophoretic pattern.

This was in agreement with results reported by many previous studies which suggested that

irradiation produces ROS that damage proteins, lipids and nucleic acid (Nair et al, 2001).

Salicin administration showed 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. The maintenance of normal protein levels after

the treatment with salicin may be due to trapping of these free radicals by this fraction, thus

preventing DNA damage.

An understanding of the tissue and organ level of antioxidant enzymes that scavenge reactive

oxygen species may provide an indication of their susceptibility to free radical-related

cytotoxic damage (Abul et al., 2002). During the present study, irradiation caused alterations

in the electrophoretic esterase pattern in serum samples. This may refer to the disturbances

occurred in the cholesterol metabolism as a result of radiation exposure. The total esterase

activities were correlated to the serum cholesterol responses in rats (Beynen et al., 1983).

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


1596


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 al. (1989). 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 radicals (El-Zayat, 2007).

The current study showed that irradiation affected electrophoretic peroxidase pattern. This

was in agreement with the study performed by Bhatia and Manda (2004) who reported that

irradiation-induced depletion in the level of reduced GSH, as well as GSH peroxidase. This

leads to elevation of the hydrogen peroxide and hence generation of the free radicals (Mills,

1960). The study showed that the decrease in GPx activity could be attributed to the

uncontrolled production of ROS and accumulation of H2O2 whereby oxidative damage to

enzymes can cause a modification of their activity (Kregel and Zhang, 2007).

Antioxidants are part of the primary cellular defense against radiationgenerated free radicals.

Radiation-induced augmentation in the levels of lipoproteins and GSH peroxidase was

significantly ameliorated by salicin treatment. The findings support property of salicin as a

free radical scavenger. This indicated the antioxidative properties of this compound against

the irradiation.

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