Evaluation Effect of the Essential Oil of Green Anis ( Pimpinella Anisum ) on The Liver Function of Young Rats Following Exposure to Mercury Chloride

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INTRODUCTION
The liver is the target organ responsible for the metabolism of xenobiotics and is therefore vulnerable to damage from chemicals.Liver damage is associated with various xenobiotics, including drugs and industrial chemicals.For this reason, many hepatoprotective agents are being studied to protect the liver from toxic attacks (Jamshidzadeh et al., 2015).
In addition, industrialization in developing countries has led to an increase in metal contamination.Heavy metals are not biodegradable and produce unwanted effects even at low doses (Mumtaz et al., 2019).Mercury (Hg) is an industrial pollutant.It induces serious alterations in human and animal tissues.Different forms of mercury compounds cause toxicity in various organs following accidental and/or occupational exposures (Hosseini et al., 2018).The liver is one of the vital and sensitive organs typically exposed to mercury toxicity (Mumtaz et al., 2019).
The objective of this study is to assess on the one hand the hepatotoxic effect of mercury on developing Wistar rats and on the other hand the hepatoprotective effect of the essential oil of green anis.

Identification of The Components of The Essential Oil by Gas Chromatography Coupled to Mass Spectrometry:
The constituents of the essential oil were identified by Gas Chromatography Coupled To Mass Spectrometry (GC/MS).Analysis was performed by Varian Saturn 2200 gas chromatography coupled to a Varian 2100 T mass spectrometer, using Saturn Varian software (Version 6, 9, 1).The capillary column was apolar with a length of 30 m, a diameter of 0.25mm, and a thickness of 0.25 μm.The flow rate of the carrier gas (helium) was 1 ml/min.1μl of essential oil was injected, using split mode (1/20).The temperature of the column was maintained at 50°C for 5 min, then increased by 5°C/min to 300°C (5 min).The detector was flame ionization (FID).The ionization energy was 70 electron volts (eV).

Animals and Treatment:
Experiments were carried out on Wistar rats (obtained from the Department of Biology, Faculty of Sciences, University of Saida) weighing 210±50 g.The animals were housed with free access to water and food in an animal room, with a 12/12-hour light/dark cycle, at 22 ± 2°C.They were mated one week after their arrival (three females and one male per cage).After one week of cohabitation with males, females were divided into 02 groups Group 1: Pregnant females received drinking distilled water.Group 2: Pregnant females received 100 mg/L of HgCl2 in distilled water (Chehimi et al., 2012).

Experimental Design:
At birth, pups issued from intoxicated females continued to receive HgCl2 in drinking water until weaning, while the control rat received only distilled water.In order to test the capacity of anise essential oil (AEO) to attenuate Hg hepatotoxicity which alteration of biochemical parameters and liver function, drug therapy was administered, beginning 24 hours after weaning.At weaning, we got 03 new groups (n = 7) as follows: Group C: Control rats (issued from control females) received distilled water only.Group Hg: received an oral solution of HgCl2.Group Hg-AEO: intoxicated rats (issued from intoxicated females) that received an intraperitoneal injection with 0,25 ml/kg of body weight AEO daily for 21 days (Asadollahpor et al., 2017).(AEO was injected pure).The number of animals that suffered was minimised in accordance with the guidelines of the European Council Directive (86/609/EEC).

Determination of Liver Parameters:
The serum samples were used to measure the activity of alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) and alkaline phosphatase (ALP) according to the methods of Reitman and Frankle (1957).

Statistical Analysis:
Results were expressed as mean ± standard error of the mean (SEM).Data were analysed by the two-way analyses of variance (ANOVAs) and Sigmastat software.When a significant difference was found, the Student-Newman-Keuls post-hoc test was conducted.For all analyses, a difference was considered significant at p ≤0.05.

RESULTS Result Of Gs/Ms:
Gas chromatographic analysis allowed us to identify 12 components for the essential oil of green anise with anethol as the major component.

Table 1: Components identified by GC/MS
The transaminase assay revealed a significant elevation in ASAT and ALAT levels in mercury-exposed animals compared to control animals (p<0.01),(P < 0.05) respectively.However, serum levels of AST and ALT in mercury-exposed animals treated with AEO showed a significant decrease compared to mercury-exposed animals (P < 0.05).
In addition, phosphatase levels showed a significant increase in mercuryintoxicated rats (p<0.01).while the levels of alkaline phosphatase in intoxicated rats treated with essential oil were significantly lower than those of rats exposed to mercury (p<0.01).

Histology:
A histological study of the liver of young rats intoxicated by mercury chloride showed that its architecture is preserved with severe vascular congestion and hypertrophy of the hepatocytes.Moreover, the liver of rats exposed to HgCl2 and treated with AEO showed vacuolar degeneration, the presence of binuclear foci, the presence of Kupffer cells and slight congestion.

DISCUSSION
The enzymatic activity of transaminases and alkaline phosphatase showed a significant increase in the batch poisoned by mercury compared to that of the control batch, which reflects hepatotoxicity due to liver injury and is explained by the leakage of these enzymes from tissue to plasma due to the alteration of membrane permeability caused by mercury, these results are in agreement with the work of Deepmala et al., (2013) and Bahi and Necib, (2015).Rats exposed to mercuric chloride show elevated levels of ASAT, ALAT and ALP evidence of its accumulation in the liver (Kumar et al., 2014).
In addition, the elevation of the plasma activity of ASAT at the mitochondrial level of hepatocytes and in the cytoplasm indicates structural damage in the liver thus causing an increase in the permeability of the membranes of the hepatocytes leading to the release of the enzyme in the liver and in the blood.Furthermore, ALAT is found in the cytosol of hepatocytes and its release from damaged cells increases its activity in plasma (Hazelhoff et al., 2018).
Elevation of ASAT indicates degradation of hepatocytes.Various studies have shown that the latter increases in the circulatory system when hepatocytes or other organs are damaged or necrotic (Nathwani et al., 2005;Mumtaz et al., 2019).
On the other hand, mercury-exposed animals treated with P.anisum essential oil have significantly reduced ASAT, ALAT and ALP levels compared to untreated exposed animals.El-Sayed et al, 2015 mentioned a hepatoprotective effect of green anise essential oil against carbon tetrachlorideinduced liver cirrhosis based on transaminase assay.Anis essential oil was able to restore serum levels of ASAT, ALAT, and ALP that were elevated following tetrachloride carbon exposure (El Sayed et al., 2015).The decrease in serum AST, ALT and ALP levels suggest that P.anisum extract could prevent liver cell damage (Cengiz et al., 2008).
Regarding the liver of young rats exposed to HgCl2, the architecture is preserved with severe vascular congestion and hypertrophy of hepatocytes.These results corroborate with the work of Hosseini et al., 2018 which confirms the presence at the hepatic level of inflammatory cell infiltration and inflammation of the central vein following exposure to a dose of 5 mg/kg of HgCl2 of which the latter increases the production of numerous ROS, such as superoxide and H2O2, which cause lipid peroxidation and subsequently oxidative tissue damage.Furthermore, the administration of HgCl2 for 21 days intraperitoneally led to severe diffuse vacuolar infection, and degeneration of hepatocytes (Abarikwu et al., 2017).Indeed, liver damage due to the introduction of mercury and cadmium is well known by higher intensities of liver enzymes in serum showing the damage caused at the level of liver tissue and the exit of enzymes into the blood (Mumtaz et al., 2019).Similarly, the results of the study conducted by Hazalhoff et al., 2018 show that female rats exhibited higher HgCl2-induced hepatotoxicity than male rats, as evidenced by greater alterations in plasma markers, liver injury, and histopathology (Hazalhoff et al., 2018).The sex-related differences observed in the liver damage produced by inorganic mercury may be explained by the greater accumulation of this metal in the liver of females.At a sublethal dose of mercuric chloride of 1.23 mg/kg treated rat tissues show liver cell damage and altered metabolic activities through the ASAT and ALAT marker enzyme pathways (Jagadeesan et al., 2014).Following HgCl2 exposure, the liver tissue was completely damaged, and degenerative and necrotic qualitative changes were observed in almost all structures of the liver tissue (Wadaan, 2009).
However, mercury-exposed animals treated with P.anisum essential oil have significantly reduced ASAT, ALAT and ALP levels compared to untreated exposed animals.El-Sayed et al., 2015 mentioned a hepatoprotective effect of green anise essential oil against carbon tetrachlorideinduced liver cirrhosis based on transaminase assay.Anise essential oil was able to restore serum levels of ASAT, ALAT, and ALP that were elevated following tetrachloride carbon exposure (El-Sayed et al., 2015).The decrease in serum AST, ALT and ALP levels suggest that P.anisum extract could prevent liver cell damage (Cengiz et al., 2008).
On the other hand, the therapy based on P.anisum EO reduced the damage of which we noticed vacuolar degeneration, the presence of binuclear foci which explains that the cells regenerate and enter a division phase, the presence of Kupffer cells which are the macrophages of the liver which indicates the presence of immune defense.
Moreover, the therapy with aqueous extract allowed the presence of peripheral lesions, foci of necrosis, normal sinusoidal capillaries and the presence of a significant number of Kupffer cells.Thus, the treatment with P.anisum including EO and aqueous extract has an almost similar effect in the face of mercury intoxication.Our results are partially different from those of Asadollahpoor et al., 2017 who found that histopathological examinations also confirmed less vacuolar degeneration and decreased macrovascular steatohepatitis in tissue sections of animals treated with high doses of P.anisum HE and aqueous extract in rats with non-alcoholic fatty liver disease.In addition, the hexanolic extract of anise seeds possesses protective effects in vitro and in vivo against carbon tetrachloride-induced hepatotoxicity, probably due to its antioxidant constituents (Jamshidzadeh et al., 2015).Also, aspartame consumption induced hepatotoxicity, and the use of P.anisum EO as an antioxidant decreased the toxicity of aspartame (El Haliem et al., 2011).

CONCLUSION
In conclusion, the results of this study show that exposure of young Wistar rats to mercury chloride during gestation and lactation period induced an increase in biomarkers of liver function as well as liver tissue damage.In addition, treatment with P. anisum essential oil reduced the level of these biomarkers and the damage that was caused by mercury.