Hazardous Effects of HighVoltage on Albino Rats and Role of Rosmarinus Ocinalis

Electromagnetic elds (EMFs) are common in our everyday lives. They have many origins and severe effects on individuals and environments where they inict a great deal of health and psychological harm. The current study investigated the impact of high voltage (H.V.) EMF 5.4 k / v for 2, 4 h per day with a frequency equal to 50 Hz on body weight(b.wt), blood indices, and certain liver enzymes of albino rats after 25 days of exposure to the electromagnetic eld. This work focuses on the therapeutic action of methanol extract of Rosmarinus ocinalis (R. ocinalis) leaves at a dose (5 mg/kg b. wt) against harmful EMF-induced effects. The ndings showed that electromagnetic eld exposure induced a substantial decrease in red blood cells (RBC), haemoglobin concentration (Hb), and catalase activity (CAT). Although white blood cells (WBCs), aspartate aminotransferase (AST), alanine aminotransferase (ALT), Total Bilirubin, Urea, Creatinine, Uric Acid, and Malondialdehyde (MDA) levels have increased signicantly under EMF treatment. Treatment with R.ocinalis showed attenuation in these parameters that were induced in rats exposed to H.V. These ndings were followed by the histopathological analysis of the liver in the observations. Finally, we conclude that R.ocinalis leaves extract offered substantial protection against H.V induced liver damage and can be applied in drug production.


Introduction
Electromagnetic elds (EMFs), a physical eld generated by moving electrically charged objects , and in uencing the actions of charged objects in the eld's vicinity, are emitted by abundant natural and human-made sources that play signi cant roles in everyday life. It stretches over space inde nitely and explains electromagnetic interaction (Emre et al. 2011). You may distinguish mainly four types of signals from EMFs. They are known as static, electrical, and MF (DC, 0 Hz), shallow frequency (ELF) elds, ranging from 1Hz to 300 Hz. An ELF-EMF symbol is the time-varying EMFs. Other technologies create intermediate frequency (IF) elds with frequencies from 300 Hz to 10 MHz and high frequency (HF) elds in the radio frequency (RF, 10 MHz-300 GHz) and microwave (MW, above 3 GHz) (ICNIRP. 2010). EMFs are present everywhere in our setting, but they are invisible to the human eye. The local build-up of electrical charges generates electric elds in the atmosphere associated with thunderstorms. The earth's MF causes the compass needle to be positioned in the north-south direction and is used by birds and sh for navigation (WHO. 2007). EMFs have several chemical impacts, such as imbalances in ion ratios, reaction to biological processes, stress and oxidative stress in the body's abundant tissues, and physiological and psychological effects on human health (Thakare and Utane 2018).
Medicinal properties produced by various medicinal plants are attributable to secondary metabolites such as avonoids, tannins, phenolic carbohydrates, glycosides, and alkaloids, which are the most signi cant bioactive plant components (Khalid et al. 2018). Rosemary (R.O cinalis L.), An aromatic plant, is a widespread household plant that belongs to the Lamiaceae family and grows in great accessories from around the world, Europe, Asia and Africa, mainly in areas surrounding the Mediterranean Sea. The bitter principle, resin, tannic acid, volatile oils, and avonoids are among the chemical constituents. It is used for the central nervous system, cardiovascular system, genito-urinary diseases, liver treatment, reproductive system and respiratory system disorders. Rosemary spices and their essential oil allow the lipid pro le, oxysterol scale, liver function, kidney function and glucose to be assumed processed. The remedy groups indicate a presumed decrease in the serum of interactive thiobarbituric acid (MDA) substances Hassanen (2015).
The current work aims to investigate the role of R.o cinalis leaves extract against EMF.

Materials And Methods
Preparation of leavesR.o cinalisextract: Leaves extract preparation: The extraction was performed separately using ethanol and methanol. The extract was prepared by stirring 100 g of plant leaves with 700 ml of solvent made up of 800 ml ethanol 95% or methanol 95% and 200 ml of distilled water on a magnetic stirrer at room temperature for 24 hours. The infusion was pumped through a piece of double-layer gauze and centrifuged for 10 min at 3000 rpm. The remainder was also re-extracted. The combined ltrates were evaporated using a rotary

Quantitative Phytochemical Analysis
Total Phenols and Total avonoids were estimated according to (Total Phenols and Total avonoids were evaluated according to (Maurya and Singh (2010)

Experimental Animals:
Sprague-Dawley strain adult male albino rats, weighing 130 ± 20 g, were obtained from Animal House, Faculty of Pharmacy, University of Al Nahda, Beni Suef. The rats were lodged in air-conditioned rooms in plastic cages at 25 ° C ± 2 (under laboratory condition). For two weeks prior to the trial, a commercially available diet and tap water were given ad libitum. All trial proceedings were performed in compliance with ethical principles veri ed by animal treatment and use guidelines of the Institutional Animal Ethics Committee, Minia University, Egypt.

Experimental Design:
After two weeks of acclimatization, forty-eight rats were divided into six groups (each 8 rats), on the following: Control group; Healthy group.
Rosemary group (R); Rats in this group were given a dose of rosemary ethanol extract ( 5 mg/kg b.wt/ daily ) for 25 days (Ghasemzadeh et al. 2011).
HV 2 h group (HV 2h); Rats in this group were exposed to two hours of high voltage daily for 25 days.
HV 4 h group (HV 4h); Rats in this group were exposed to four hours of high voltage daily for 25 days.

Blood samples
At the end of 30 days, rats were fasted overnight and anesthetized to collect the blood samples from the retro-orbital plexus (Schermer 1967). For hematological tests, appropriate volumes of fresh blood were taken directly into the heparinized tube. The other blood sample accessories were allowed to coagulate at room temperature, then the clear non-haemolysed sera at 4 o C were isolated and stored at -20 o C before biochemical analysis was used.

Tissues samples
Rats were sacri ced, excised the liver cleaned with philter paper. Small liver accessories were set for histopathological tests in 10 percent formalin solution. Other liver tissue accessories were homogenized and used to assess the concentration of CAT and MDA.
Globulin was certain by the difference between total protein and albumin. Total and direct bilirubin were certain calorimetrically (Walters and Gerarde 1970), using enzymatic colorimetric proceedings Kits from Bio-Diagnostic Co., Egypt.

Measuring of liver CAT and MDA
Half a grams of liver tissue from each animal is homogenized in 5 ml of 100 mM phosphate buffer, pH 7.4 on ice using Universal laboratory Help homogenizer, and then centrifuged at 3000 rpm 4oC for 15 min. The supernatants of the creatures were composed and stored at -20oC until the concentration of catalase vigor (CAT) and malondialdehyde ( MDA) was calculated in the manner mentioned in Aebi (1984) and Ohkawa et al. (1979).

Histopathological examination:
Specimens of liver were set in 10 percent formalin solution. It was cut into 5 cm thick sections after 24 h of xation accompanied by embedding in a para n block and stained with hematoxylin-eosin (H&E) for routine histopathological analysis according to Bancroft et al. (1996).

Statistical analysis:
The results gained in this work were evaluated by the SPSS Statistics One Way ANOVA test. The results were expressed as mean ± standard error, and values of P < 0.05 were considered statistically presumed (Snedecor and Cochran 1986).

Results And Discussion
Qualitative Analysis of Phytochemicals: Phytochemical ndings in (Table 1) suggest the presence of phytochemicals in R.o cinalis leaves such as steroids, saponins, tannins, anthocyanins, emodins, avonoids, terpenoids, glycosides and alkaloids and phenols in two extracts. In contrast, fatty acids were absent in ethanol and methanol extracts, whereas emodins and alkaloids were absent in methanol extract.
This data agree with Edrah et al. (2017), who reveal that the R.o cinalis ethanolic extract contains avonoids, terpenoids, glycosides, and phenols, whereas saponins have been absent. Also, in the same line with Khamis and Aly (2017), who found that terpenoids, alkaloids, glycosides, saponins, and avonoids were widely present in ethanolic extract R.o cinalis. These plants were used as preservatives for food and as folk medicines. Terpenoids are used in cough, asthma, and hay fever treatments.
Saponins have antibiotic properties and guard against hypercholesterolemia (Mir et al. 2013).  Table 2. Phenolic compounds are one of the primary, secondary metabolites that protect against oxidative stress since they can act as reducing agents through single-oxygen scavengers and donors of hydrogen atoms with subsequent stabilization of the generated free radicals that form stable compounds that do not initiate or propagate oxidation (Karthivashan et al. 2013).
Previous studies manifest the relationship between the total avonoid content and antioxidant vigor of medicinal plants (Kaur and Mondal 2014). Flavonoids commonly contain anthocyanins, avanols, avones, avanones, and avonols as the polyphenols. The difference in solvent polarity and diffusion forces, the structural complexity, or the selective solubility of phytochemicals in a given solvent may explain the distinction between phenolic and avonoid content and the extraction solvent (Medini et al. 2014). Table 2 Total phenolic compounds and total avonoids ofR.o cinalisExtracts * Total phenolic compounds (mg/g) a Total avonoids Rosemary Rosmiry Methanol extract 5.40 ± 0.14** 6.54 ± 0.023*** Ethanol extract 6.67 ± 0.14*** 5.90 ± 0.045** *a: mg GAE /g of dryleafes extract; b: mg QE/g of dryleafes extract. Each value is expressed as the mean.± SE (n = 3). (**and ***) are Signi cant and highly signi cant respectively at P < 0.05 .

Antioxidant Activity
The DPPH is a stable free radical at room temperature and accepts an electron/hydrogen radical to become a stable diamagnetic molecule (David et al. 2004). The DPPH is usually used as a substrate to evaluate the antioxidant vigor (Edamatsu et al. 1989). The antioxidants react with the stable free radical DPPH and turn into it to 1,1-diphenyl-2-picryl hydrazine with decoloration (Kumar et al. 2012). DPPH radical's reduction potential is certain by the dwindling in its 517 nm absorbance, which is caused by antioxidants. Visually visible, like a change in color from purple to yellow. The latest ndings for R.o cinalis extract manifest free radical scavenging vigor (  Effect of R .o cinalis extract on hematological parameters in blood rats exposed to HV 2 and 4 h. In this study the hematological tests of the animals treated for 30 days with the ethanolic extract R.o cinalis were carried out. Table ( 1) shows no presumed divergence in the scale of Hb, WBCs, and RBCs relative to the control. It was estimated that Hb (P < 0.05) decreased by around 15 and 11 % in the exposed HV groups for 2 or 4 h, compared with the control group, respectively. In the meantime, remedy groups with ethanolic R.o cinalis extract increased probably (P < 0.05) this reduction of Hb and restored it to normal control. RBCs (P < 0.05) were assumed to decrease by about 12 and 6 % respectively in the exposed group to HV for 2 or 4 h compared to the control group. Meanwhile, preremdy groups with ethanolic R.o cinalis extract improved presumed and returned RBCs to normal control. WBCs (P < 0.05) were assumed to increase by about 22 percent and 54 percent respectively in the exposed group to HV for 2 or 4 h compared to the control group. Packed cell volume (PCV) was thought to decrease by about 24 % and 30 % in the exposed group to HV for 2 or 4 h, respectively, compared to the control group. In the meantime, preremdy groups with ethanolic extract of R.o cinalis strengthened presumed PCV and took it back to normal regulation. The RBCs are assumed to dwindle (P < 0.05) by around 12 % and 6 % relative to the control group in the exposed group to HV for 2 or 4 h, respectively. In the meantime, presumed preremdy groups with ethanolic extract of R.o cinalis strengthened and returned RBCs to normal regulation. WBCs are assumed (P < 0.05) to increase by around 22 % and 54 %, respectively, in the exposed group to HV for 2 or 4 h relative to the control group. Meanwhile, it was thought that preremdy groups with ethanolic R.o cinalis extract (P < 0.05) decreased this increase in WBCs by around 9 % compared to the 2 h exposed group to HV. In contrast, this re ning was 25 % compared to the 4 h exposed group to HV.
Xiao-Feng and Gun con rm the current data (2017) 's data, who found the elevation of white blood cells in rats exposed to HVTL and radiation exposure (Hsu et al. 2010). Also, in harmony with about hematological parameters (Eid et al. 2015). The depletion in the values of hematological parameters following EMF radiation may be attributed to direct deterioration caused by radiation and overproduction of ROS by microwave radiation interaction, causing hemolysis (Aweda et al. 2004). The dwindling of RBCs may be due to the interaction between heme (iron) and SMF, where the magnetic eld penetrates the body and acts ions in all organs, altering the cell membrane's potential disrupting the ions (Kula and Drozds 1996). Although IB serum scale was assumed to diminish (P < 0.05), handling with either R.o cinalis extract was simultaneously with 2 or 4 h HV treatment and restored to almost control. With accordance to these data Salem et al. ( 2005) postulated that the damaging impactof static magnetic eld on the liver is manifested by an raise in blood AST and the most speci c marker of liver cell deterioration ALT vigor. The exposure to ELF-EMF of 2 mT and 50Hz raised presumed serum transaminases vigor in mice. Hashem and El-Sharkawy (2009). ALT and AST vigor improved presumed (P<0.05) for two months in serum and liver tissue homogeneous to EMF exposed rats in the same manner as standard control group Eid et al.   Table (3) indicate no presumed difference in the 30-day serum protein and albumin scale between R.o cinalis extract remedy groups compared with the control group. Meanwhile, HV exposure for 2 or 4 h increased the serum protein scale by around 39.5% and 43.1%, respectively (p > 0.05), while the albumin scale increased by 58% and 75% compared to the control group.
In a group remedy with R.o cinalis, the serum protein scale was assumed to dwindle by around 20 % simultaneously with HV treatment for 2 or 4 h. In conjunction with exposure to HV for 2 or 4 h, R.o cinalis also decreased albumin by around 17 % compared with HV for groups of 2 or 4 hours. These results are in agreement with those data reported by plentiful authors (Hashem and El-Sharkawy 2009). The increase in the concentration of serum protein and albumin were observed as a result of deteriorating cells that ow into circulation after exposure to the magnetic eld (Ibrahim et al. 2008). Kulkarni and Gandhare (2015) found that total protein, albumin, and globulin display a transient 30-hour increase that decreases at 60 hours relative to the regulated community. The period of intensity and exposure of EFs has been found to play a vital role in inducing internal elds and activating biological differentiation.   The urea scale decreased by approximately 14 % after 4h relative to the group exposed to HV for 4h. On the other hand, creatinine was assumed to be reduced by about 32 %, and 36 % by R.o cinalis extract simultaneously as HV exposure (p>0.05) compared with groups exposed to HV 2 or 4 h, respectively.
Also, R.o cinalis extracts reduced uric acid by around 22 % simultaneously with the assumed exposure to HV (p>0.05). The current ndings are consistent with Sharma et al. (2017). In rats exposed to electromagnetic radiation, those who reported serum uric acid, urea, creatinine (p>0.05) were presumed to have increased. Irradiation can cause DNA molecules to split, and their bases (purines) broken, which can be catabolized into uric acid (Ganong 1999). Creatinine is predominantly produced in the muscles, and freely appears in the blood plasma and urine. Also, a reported rise in serum urea (p < 0.05) and creatinine due to sensitivity to extremely low-frequency electrical elds (Kulkarni and Gandhare 2015) and exposure to EMF of 2 mT, 50Hz (Hashem and El-Sharkawy 2009).
Effect of R.o cinalis extract on oxidative stress in liver tissue homogenate of rats treated with HV: ROS formation may be derived from oxidized dietary fats. It may lead to increased protein deterioration in the liver by enhancing cell membrane LPO. Increasing ROS generation can lead to calcium homeostasis disorders, increased membrane uidity, and cell death (Hassanen 2015).  Effect of R.o cinalis extract on histological examination of rats exposed to HV for 2 and 4 h Histological changes to help the tested biochemical markers of damage to organs were screened. It is noted that autopsy samples taken from rat liver exposed to HV for 2 or 4 h manifest several alterations such as activation of kupffer cells, central vein and sinusoid swelling, hydropic hepatocyte degeneration, and broplasia in a portal triad with the emergence of focal hepatic necrosis associated with in ammatory cell in ltration in a community exposed to HV for 4 h. However, handling with either R.o cinalis improved hepatic histopathology, with only mild kupffer cell activation in the group exposed to HV for 2 and 4 h. concomitant with R. o cinalis. (Table 6 & Fig. 1).
The ELF-EMF caused focal centrilobular necrosis of the hepatic cells surrounded by severe hydropic degeneration involving most hepatic parenchyma in liver mice (Hashem and El-Sharkawy 2009). Table 9 Histopathological notes on liver tissue of rats exposed to HV for 2 and 4 h plus R.o cinalis.