Phytochemical Screening of Cinnamon Cassia and Its Protective Effects Against Hepatotoxicity Induced By Difenoconazole in Male Albino Rats.

The objective of the present study has focused on the phytochemical analysis of Cinnamon cassia bark for determination of bioactive components, which have been associated with antioxidative stress induced by difenoconazole treatment in hepatic tissue of male albino rats. Ninety rats were assigned randomly to 9 groups, each group comprised of 10 animals. The rst group served as control animals were administrated distilled water and the rest served as the experimental groups. Groups II and III animals were orally administrated with difenoconazole at doses of 58.9 and 117.8 mg/kg BW (represent of 1/20 and 1/10 of oral LD 50 , respectively) while the groups IV and V animals were received aqueous extract of cinnamon (AEC) at doses of 200 and 400 mg/kg BW , respectively . In addition groups VI and VII, animals were received AEC prior to 2h of administration with difenoconazole at low dose as well as groups VIII and IX, rats were received with AEC before treatments with high dose of difenoconazole for 28 days. Results of the present study indicated the presence of total phenolic, avinoids and tannins as the main bioactive components in the AEC. Furthermore, the nal body weight and liver index were increased markedly in difenoconazole-treated rats and these parameters values were comparable to control group following co-administration with AEC. However, difenoconazole-treatment induced a signicant elevation in the level of (LPO) associated with adepletion of GSH level and an elevation in the activities of serum liver enzyme markers (i.e., AST ,ALT,ALP and GGT) was observed. These results conrmed with histopatlogical ndings . In contrast, treatment with AEC in difenoconazole-treated rats elevated the level of endogenous hepatic antioxidant system (SOD, CAT and GSH) along with reducing the activities of serum liver enzymes. However, the hepatic protective property of AEC was further conrmed by histopathological ndings. These ndings may be attributed to the presence of total phenolic , and tannins , which have anti-oxidative effect against oxidative injury- induced by tested fungicide.


Introduction
Pesticide are known to induce oxidative stress by production of reactive oxygen species (POS) besides releasing of highly reactive metabolites (Rastogi et al ., 2009).
The triazole is chemical family of fungicides, introduced in the 1980s, and it is the most important classes of pesticides in agriculture. In addition, they have the excellent protective and curative properties against a wide rang spectrum of crop diseases, where they are commonly used as environmental fungicides for grain, vegetable, fruits and owers production world wide (Wang et al., 2011).
These triazole fungicide consists of numerous members such as difenoconazole, propicanazole, tridimofen and fenbucanazole .and they are designed to inhibit the activity of lanosterol 14α-demethylase (CYP51), a key enzyme for ergosterol biosynthesis in fungi, causing membrane dysfunction (Chaâbane et al., 2016).
Difenoconazole is abroad spectrum fungicide belong to the triazole group of fungicides, and it used to control a broad spectrum of foliar, seed and soil-borne diseases.
By the oral route of exposure, these triazole would be considered as having low to moderate mammalian toxicology (Costa, 1997).
Recently it is suspected that long -term exposure to difenoconazole via drinking water and food may induce hepatotoxicity and tumorigenesis (Wang et al .,2011).
Abd-Alrahman et al. (2014) studied the effect of oral administration of difenoconazole at a dose of 10mg/kg to rats for 28 days. They found a signi cant increase in serum alanine aminotransferase (ALT), gamma-glutamyl transferase All animals were observed for clinical signs of toxicity and the mortality was recorded within the experimental period.
However, individual body weight was measured at the initiation of experimental and weekly during the treatment period (28 days).The study was conducted in compliance with [OECD guidlines ,No, 407.2008].
Collection of blood and tissue samples.
At the end of the experimental, rats were fasted overnight and weighted and the blood samples from each rat was collected by orbital sinus vein (Stone, 1954) in sterilized dry tubes and kept to coagulation for 30 min at room temperature. The clear sera obtained after centrifuged at 3000 rpm for 15 min and then stored at -20°C till biochemical analysis.
Afterthat, animals were sacri ced by cervical dislocation on day 29, the liver of each rat was collected and divided into two portions. Small portion were xed in neutral bufferd formalin (10%).for histopathological examination (Banchroft et al., 1996).The other portion of liver was washed with ice-cold saline ,blotted dry ,then wrapped in foil and immediately placed in liquid nitrogen and stored at -80°C for subsequent measurements.
Liver weight and liver weight index.
Fresh liver from the treated rats will blotted dry and subsequently weighted. Absolute liver weights will be recorded and the change in liver weight were recorded relative to body weight.

Sero -biochemical measurments :-
The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured according to the method of Reitman and Frankel (1957), alkaline phosphatase.was determined according to the method of Klein et al. (1960) and gamma glutamyl transferase (GGT) was determined according to the method of Szasz and Persijng (1974).While the total protein and albumin concentrations were determined according to the method of Henry (1974) and Young (1975), respectively. However, the globulin concentration was calculated mathematically according to Coles (1986). All kits obtained from sigma chemical Co.

Determination of hepatic Oxidative Stress markers:
One portion of liver was washed in physiological saline, and then homogenized and the homogenate used for estimation of MDA and GSH levels as well as the activites of GGT, SOD and CAT.
Malondialdehyde (MDA) is an end-product of membrane lipid peroxidation (LPO) and the enhanced production of MDA in tissue is an index of oxidative stress. Therefore, MDA concentration was estimated by the method of Ohkawa et al. (1979). The level of lipid peroxides is expressed as n mol MDA /g tissue.
Determination of hepatic reduced glutathione (GSH) content.
Reduced glutathione content was determined in the hepatic tissue according to the method of Ellman (1959) the level of GSH was expressed as mg/g tissue.
Superoxide dismutase (SOD) activity was assayed in liver tissue homogenate by the method of Marklund & Marklund (1974). This method is based on the auto-oxidation of pyrogallol,. The pyrogallol auto oxidation is highly dependant on O2 and is inhibited by the presence of SOD. The SOD activity was determined spectrometrically at 440nm and expressed as U /mg protein.
Determination of Catalase (CAT) activity.
Catalase activity was determined according to the method of Cohen et al. (1970). By measuring the decomposition of hydrogen peroxide (H2O2) at 240 nm and expressed as U /mg protein.
Gamm -glutamyl transferase (GGT) activity was determined in liver tissue homogenates according to the method of Szasz and Persijng (1974 ).
The rate of colored liberation of yellow indicator 5-amino-2-nitrobenzoat is directly proportional to GGT activity in the sample and is quantitated by measuring the increase in absorbance at 405 nm.
Determination of tissue protein.
The level of protein in liver tissue was determined according to the method of Bradford (1976) .values were expressed as mg/g of tissue.
Liver tissue samples intended for the histopathological investigation were xed in 10% neutral formalin, prepared and stained according to Bancroft et al.(1996).The degree of damage was graded as follow as mild (+),moderate (++)and severe (+++)damage .

Statistical analysis.
All results are expressed as (mean ± SE). Within groups comparisons were preformed by the analysis of variance using the ANOVA Test .Signi cant differences between the control and the experimental groups were assessed by the Student T Test .The result were considered signi cant at P < 0.05 .all statistical analysis were preformed using SPSS( statistical package for social sciences ).

Results
Phytochemical screning of Cinnamon cassia.
The results presented in Table (1) showed that the aqueous extract of Cinnamon (AEC) had the highest content of total phenolic (31.17 mg GAE/g) followed by avenoids content (18.58 mg GAE /g), and Tannis (10.14 mg GAE/g). Changes in body weights and liver index. Table (2) summarized the effect of difenoconazole alone and co-administration with aqueous extract of cinnamon (AEC) on body weight and liver index throughout the experimental period (28day).
Difenoconazole treatments for 28 days, caused a slightly signi cant increase in the nal body weight (by 3.8 and 5.7%, respectively) compared to control group. As shown in Table (2) with regard to liver index, a signi cant increase in the absolute and relative weights of liver was observed in rats treated with difenoconazole in compared with control group.
Supplemented rats with AEC alone did not induce any signi cant change in the nal of body weight and liver index when compared with control group as presented in Table (2). However, administration of AEC pre-treatment with difenoconazole resulted in insigni cant decrease in the nal body weight and liver index was observed when compared with difenocanazole-treated rats. Interestingly, no signs of toxicity in the experimental, and no mortality was recorded. Data between treated groups were analyzed using (one way ANOVA). (a) signi cant compared to control.
(+) increase compared to control group.
(-) decrease compared to control group.
Changes in serum hepato-speci c markers.
The extent of hepatic injury assessment by determining the serum liver enzyme markers, i.e, ALT, AST, ALP and GGT in blood circulation as well as the concentrations of serum total protein, albumin and globulin were determined.  (Table 3).
As shown in Table (3), after 28 days of AEC supplementation to male albino rats, there was no signi cant differences between the control group and each treatment group in the activities of ALT, AST, ALP and GGT.
The results presents in Table (3) revealed that co-administration of AEC, ameliorated the activities of serum ALT, AST, ALP and GGT, which are signi cantly lower than values of difenoconazole -treated rats and become near to normal values of control group.
However, administration rats with difenoconazole at high dose produced a signi cant elevation in the level of total protein (by 17.39%) and insigni cant increase (by 11.43%) in this parameter was observed in rats treated with low dose of difenoconazole in compared with control group.
On the other hand, the albumin concentration did not change markedly in rats treated with difenoconazole throughout the experimental period. In contrast, Cinnamon treatments did not induce any signi cant alteration in the levels of TP, albumin and globulin within the experimental period.
Co-administration of AEC led to a signi cant decrease in the levels of TP and globulin in rats treated with low dose of difenoconazole in compared with difenocanazole-treated rats.
Also, a signi cant decrease in the level of TP was noticed in rats supplemental with high dose of AEC prior to treatment with high dose of difenoconazole when compared with difenoconazole-treated rats. Data between treated groups were analyzed using (one way ANOVA). Concerning the effect of difenoconazole-treatment on the hepatic lipid peroxidation, the level of MDA in hepatic tissue of difenoconazole -treated rats was signi cantly elevated (by 29.13 and 34.47%) when compared to the level of MDA in control group (Table 5).
With regard to reduced glutathione level (GSH), there was a signi cant decline (by32.17 and 35.08%, respectively) in the glutathione level in hepatic tissue of rats treated with both doses of difenoconazole, respectively in compared with control group as shown in Table 5 .
On the other hand, the enhancement in the activity of GGT was observed in rat treated with difenoconazole in compared with control group.
Furthermore, as seen in Supplemented rats with AEC did not cause any signi cant alteration in the levels of MDA and GSH as well as in the activities of GGT and CAT, whereas a signi cant elevation in the activity of SOD (by39.7 and 43.47%) was detected in compared with control group ( Table 5).
Administration of AEC before treatment with difenoconazole resulted in reversing difenoconazole induced increase in MDA and decrease in GSH level when compared with difenoconazole-treated rats.
Supplementation rats with AEC prior to treatment with difenoconazole at low dose caused a signi cant decrease in the level of MDA and increase in the level of GSH whereas, a signi cant reduced in the activity of GGT was noticed in rats treated with difenoconazol after supplementation with AEC in compared with difenoconazole-treated rats .In addition, a signi cant increase in the activities of SOD and CAT was detected in rats treated with high dose of difenoconazole following administration of AEC in comparison with difenoconazole-treated rats.
But Cinnamon treatments markedly improved GSH level in compared to the difenoconazole treated rats especially at low dose of tested fungicide.
The results presented in table (5) illustrated that rats received AEC at high dose, pre-treatment with low dose of difenoconazole had a signi cant decrease in the levels of MDA and GGT activity, whereas a signi cant elevation in the level of GSH and the activities of SOD and CAT was noticed in compared with difenoconazole-treatment rats.
Rats treated with high dose of difenoconazole after received the AEC had a signi cant decrease in the level of MDA and GGT activity ,whereas a signi cant enhancement in the activities of SOD and CAT was detected in compared with difenoconazole-treated rats(at high dose ). In contrast, the GSH level did not alter markedly in compared with difenoconazole-treated rats. Histopathological ndings.
The histological changes in treated rats are graded and summarized in Table (6). The histopathological examination of the liver sections of the control group, showed normal histogical structure Fig 1(A).
The most prominent histopathological ndings were observed in the liver of difenoconazole-treatment rats were degenerative changes in the hepatocytes surrounded and adjacent the dilated central vein of rats following treatment with high dose of difenoconazole for 28 days, in addition, the portal area showed oedema and periductal brosis surrounding the bile duct, as well as hyperplasia in other bile ducts was detected Fig 1(B Liver of AEC supplemented rats showed a normal histological structure Fig 1(H) with slight degenerative change especially at high dosage level and congestion in rats received the AEC during the experimental period.
When compared to difenoconazole-treated rats, rats received AEC pre-treatment with difenoconazole showed improvement in the histology of liver as shown in the Fig 1(I, J).  Recently, considerable attention has been focused on medicinal plants, which play a vital role for medication and to determine the active principles responsible for protecting or preventing the development of oxidative stress, which is an important cause of many chronic diseases.
Therefore, the present study carried to evaluate the adverse effects of difenoconazole fungicide and attempt of the possible anti-hepatotoxic of Cinnamon cassia against difenoconazole induced hepatotoxicity in male rats.
Phytochemical Screening of Cinnamon cassia.
The phytochemical investigation of Cinnamon cassia revealed the presence of total phenolic, avonoids and tannis as shown in Table (1). Cinnamon is known to have contain of phenolic compound and avonoids which act as potent antioxidants due to their ability to scavenge free radical and form relatively inert phenoxy radical intermediates [Ferguson, 2001].
The phenolic compounds ,such as avonoids ,phenolic acid and tannins posses divers biological activities , and they are responsible for antioxidant action as well as a potent scavenger of hydrogen peroxide( H2O2),nitric oxide (NO)and lipid peroxide free radicals [Aravid et al .,2012].
In addition ,the antioxidant activities of phenolic compounds is mainly due to their redox properties such as adsorbing and neutralizing free radicals as well as quenching single and triple oxygen or decomposing peroxides [Yashin et al .,2017].
With regard to avonoids , they are typical phenolic compounds, which in turn ,act as potent metal-chelators and free radical scavengers.
Generally, they have higher antioxidant activity against peroxy radical than phenolic acid due to multiple hydroxyl There was a signi cant decrease in the levels of T4 and T3 in rats treated with difenoconazole for 28 days (data not shown).
In addition, difenoconazole could be induce accelerated differentiation of immature adipocytes into mature cells and may disrupt the lipid metabolism of difenoconazole-treatment rats [EFSAb]. Concerning the liver weights when expressed as absolute or relative to body weight ,there was a signi cantly increase liver weight (hepatomegaly) in rats following expouse to difenoconazole. This could be explain by the induction of several xenobiotics metabolizing enzyme (FFSAb, 2008).
A hepatomegaly often accompany with increase in hepatic -derived enzymes (i.e ., Transaminase ,ALP and GGT),that may appear in the plasma following liver enlargement Meanwhile, the absolute and relative weight of liver rats supplemented only with AEC comparable to control and no signi cant differences were found in their values .
However, it was observed that co-administration of AEC with difenoconazole, attenuated the increase weight of the liver following treatment with difenoconazole and consequently caused recovery towards normalization comparable to the control group. This indicated that AEC could exert in vivo hepatoprotective effect.
Previous study have shown that cinnamon zeylanicum treatment reduced the relative weight of the liver [Shan et al .,1998].
Changes in Serum hepato -speci c markers.
Assessment of liver injury by difenoconazole is usually made by determination of serum enzymes level of ALT, AST, ALP and GGT, which are important markers to evaluate hepato-billary injuries.
In the current ,an increase signi cantly in the activities of serum ALT and AST was observed following treatment with difenoconazole, which have been attributed to leakage of cellular enzymes in circulation as a result of loss function integrity of cell membrane of hypatocytes [ Navaro et al .,1993 andOzer et al .,2008] A number of chemical including agro-pesticides and various environmental pollutanta can severe cellular damage in different organs in the body .This occurred through metabolic activation to highly reactive substances such as free radicals (Satpute et al., 2017).
In addition, there was also a signi cant elevation in the activities of ALP and GGT in difenoconazole treated rats, this induction of ALP, may be a attributed to presence of increasing of billiary pressure rather than increased release from damage cells .and this elevation may be occurred during liver regeneration [Moss and butterworth , 1974]. However, enhancement of ALP activity may be due to elevation of bile acids concentration during cholestasis is apparently necessary for the release and transport of solubilized hepatic ALP to serum.
Also, an induction in the activity of GGT was detected in rats treated with difenoconazole within the experimental period.
ALP and GGT activities are important biomarkers to evaluate hepatobilliary injuries or bile duct lesion in the rat liver, where ALP is primary marker of hepato-biliary effects and cholestasis (moderate to marked elevation (Ramaiah , 2007) GGT is a cell surface enzyme that cleaves Y-glutamyl bonds (Godwin et al 1992).It is localized to the luminal surface of ducts and glands through out the body (Hanigan . 2014).

However, GGT is abiliary enzyme that is especially useful in the diagnosis of intra-hepatic cholestasis and obstructive
Jaundice (Stein et al ., 1989).
In addition, GGT is more responsive to biliary obstruction than alkaline phosphatase , where GGT has no origin in bone or placenta ,and hence, GGT is not increased in bone disorders as is ALP .
In rats, GGT activity is considered a reliable marker for cholestasis compared to alkaline phosphatase activity (Ozer et al ., 2008). In addition, the rat GGT assay detects bile duct hyperplasia and necrosis [Leonard et al ., 1984].
The biochemical markers in the liver viz., ALT, AST and ALP were signi cantly elevated in rats treated with It is worth mentioning that C.cassia was able to ameliorate all the altered hepatic biomarkers post-treatment with difenoconazole ,this suggest that the vital role of Cinnamon extract in protecting the integrity and the function of cells and tissue .
Co-administration cinnamon with difenoconozole showed signi cant decrement in the activities of ALT ,AST,ALP and GGT compared to difenoconazole-treated rats , this indicate that the extract may reduce hepatocellular damage.
Co-administration of cinnamon extract in doses of 200 and 400mg/kg B.W, attenuated the increased levels of serum enzymes[i.e., ALT,AST and ALP] produced by difenoconazole and caused subsequent recovery towards normalization comparable to the control group .this in turn ,indicate that AEC could reduce liver injury -induced by difenoconazole . Hence, cinnamon extract its hepatoprotective effects as a result of presence of wide range of bioactive compounds including phenolics.
Cinnamon extract at doses of 200 and 400mg/kg B.W when orally given to obese diabetic rats ,signi cantly lowered the high serum levels of ALT,AST and ALP enzymes ,when compared to the positive control groups [Shalaby and Saifan ,2014].
The hepatoprotective effect of Cinnamon extract was evident from the signi cant decrease in serum activities of liver enzymes (ALT,AST&ALP)in obese diabetic rats [Shatwan et al .,2013].
Moselhy and Junbi (2010) observed that the elevated serum AST and ALT activities induced by CCL4, were restored towards normalization signi cantly by orally administration of 200mg/kg cinnamaldehyde (CNN) once a daily for 7 days when compared to control rats.
Our biochemical nding were con rmed with histopathological ndings of rat liver sections in the current study.
An elevation in the serum total protein concentration and globulin levels was observed in rat treated with high dosage level of difenoconazole, whereas no signi cant differences in the concentration of albumin.
In addition, the albumin /globulin ratio (A/G ratio) was signi cantly decreased in the groups of rats treated with difenoconazole .This could be attributed to increase of serum globulin level as the result of the immune response of toxic effect.
Meanwhile, in the present study co-administration of AEC lowered the levels of TP and globulin and their values were found comparable to control values.
Changes in the markers of oxidative stress and antioxidant status .
It is well-established that the liver is a target organ for xenobiotics substances and it is play crucial role in the detoxi cation process. Therefore, any injury or impairment of its function produces hepatotoxicity on living organism.
Oxygen free radical (superoxide, O2.) and hydroxyl radical (OH.)and hydrogen peroxide (H2O2) called reactive oxygen species, which play a signi cant role in oxidative stress and they are also capable presenting a toxic action on self tissues causing lipid peroxidation [Sharmanov et al .,1990 andBaxter et al .,1983].
The elevation of oxidative stress can be monitored by several markers .The monlondialdehy (MDA) measure is considered as an indicator of lipid peroxidation (LPO) [De-Zwart et al ,. 1990].
Di noconazole-treated induced lipid peroxidation in hepatic tissue, which observed by marked elevation in the level of MDA, this suggest that it mediated free radical induced lipid peroxidation in hepatocytes was strongly associated with tissue injuries.
In the present study, enhancement of MDA level in liver homogenate, suggest enhanced LPO leading to increase the preambility of the cell membrane of hepatocytes, consequently release of transaminases (ALT, AST) into circulation as shown in our result.
Therefore, the remarkable elevation of ALT and MDA in the current study supported that difenoconazole mediated lipid peroxidation in liver rat as a result of reducing the antioxidant potential and hence accelerating the oxidative damage of hepatocyte.
The MDA is by-product of lipid peroxidation ,while the glutathione (GSH)content could be used to evaluate the antioxidant status of cellular system [Dall-Donn et al .,2007,Circu and Aw,2008and Pallarda et al ., 2009].
The glutathione (GSH) is non-enzymatic antioxidant and it is one of the most abundant tri-peptide and it present in the liver and its essential function are mainly concerned with maintain structure and function integrity of cell via removal of free radical. Therefore, it playa vital role in production against oxidative stress [Halliwell and Gutterdge 1990].
Our results revealed that difenoconazole treatment induce a signi cant deplection of GSH content in hepatic tissue and production of antioxidant enzymes viz , SOD and CAT.
In contrast, there was a signi cant elevation in the activity of hepatic GGT in difenoconazole -treated rats.
Induction of GGT expression it is considered as an adaptive response or as part of natural hepatocyte protective mechanism for GSH turnover (repletion) in hepatic tissue .The body's antioxidant defeneces undergo consumptive depletion following oxidative injury. Therefore, low antioxidant defense status are also correlated with elevated GGT, particularly reduced levels of GSH. However, GGT is needed to metabolize glutathionylated xenobiotics in liver and hence its elevation association with increased exposure to xenobiotics [Koening and Seneff ,2015].
Previous studies have shown that usually the deleterious effects of oxidative stress are counteracted by endogenous antioxidative enzymes, which provide a major defensive mechanism against free radical damage .The most important antioxidative enzymes i.e ., SOD and CAT, when ROS begin to accumulate in the cells, exhibits defensive mechanism using various antioxidant enzymes .The main detoxifying system for peroxides are CAT and GSH However, superoxide dismutase (SOD), a common enzyme play an important protective role by catalyzing the removal of superoxide radical (O2.), and converted to hydrogen peroxide (H2O2) and hence (H2O2) is degraded by catalase (CAT) which catalyzes the reaction between two hydrogen peroxide molecules (H2O2) and this reaction results in water and O2 production and prevent form a highly reactive OH in presence of Iron as catalyst [Turner and Lysiad ,2008]. Our ndings reveled that SOD activity was decreased in the hepatic tissue .The reason of the decreased SOD could be attributed to excessive ROS generation [Scott et al ., 2008].
Also this decrease could be due to a feedback inhibition or oxidative inactivation of enzyme protein due to excess ROS formation .
However, the reduction of the activity of CAT re ect inability of hepatic tissue to eliminate H2O2 produced the activity of CYP and inactivation of this enzyme as a result of excess of ROS production and /or suppressing of heme biosynthes [Pigeolet et al ., 1990].
Recently, considerable attention has been focused on some medicinal plants like cinnamon which are posses diverse biological activities.
The total phenolic might be the main bio-active compounds which, a potent scavenger of hydrogen peroxide( H2O2),nitric oxide (NO)and lipid peroxide [Aravind et al .,2012].The antioxidant activities of phenolic compounds is due to their redox properties.
In addition, the phenolic hydroxyl groups is shown to denote electron to oxygen radicals and also reduce ferrious ion to ferric ion, thereby suppressing the oxidation [Morel et al .,1993].
However ,it is found that cinnamon contain proteins ,carbohydrates .vitamins (A,C,K and B3 ) and minerals like The hepatoprotective potential of Cinnamon cassia against the oxidative stress induced by di noconazole was evaluated in this study.
Apparently, the intracellular defenses based on glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) are insu cient to inhibit these pro-oxidant reaction, but the nature antioxidant supplementation impart protection against oxidative stress, via increased the intracellular antioxidant defenses,which can be overwhelmed .
In the current study, remarkable elevation of SOD and CAT activities was observed following co-administration of AEC with difenoconazole.
It is probable that initial increase in hepatic SOD and CAT level was occurred as adapative nature of the defense system against damage effect of superoxide radicals (O2.) in liver brought about by difenoconazole treatment.
Since, polyphenol effectively removes oxygen radicals, and diminished degradation or inactivation of antioxidants The major histopathlogical nding were observed in the liver of rats treated with high dose of difenoconazole as shown in Table ( 6) and Fig (1).
These ndings might further con rm that hepatic injury induced by difenoconazole is associated with generation of free-oxygen radicals and the biochemical results supported these nding. The cell injury cause the release of cytokines, especially Tumor Necrosis factor-alpha [TNF-α] which generate ROS from the tissues, and consequently produce lipid peroxidation [Lachleitner et al .,2000].
The degeneration condition observed in liver of difenoconazole-treated rats correlated with the detected biochemical alteration wherein, an increase in the level of ALT, AST, ALP , GGT and LPO was noticed .
However, histopathological observation in AEC-supplemented rats con rmed with biochemical results showing the normal cellular architecture ,in particular low dose of AEC (200mg /kg B.W) this in turn ,substantiate the safety pro le of the cinnamon extract at low dosage level.
In addition, our result revealed that rats received cinnamon extract prior to difenoconazole-treatment showed improvements in the histology of hypatocytes in comparison to difenoconazole-treated rats. This suggest that cinnamon has a role in counteracting the oxidative damage-induced by generation of free radicals via difenoconazole treatment.
Indeed, administration rats with low dose of AEC did not induce any degenerative effect, although this extract at high dose (400mg/kg B.W) showed degenerative change.
In such cases, low dose of AEC could not damage mitochondria and antioxidant compounds was not oxidized and could scavenge free radicals.
In contrast, high dose level of AEC could damage and permeablize and could react as pro-oxidant damaging rat hepatocytes. It seems that switch from anti-proxidant reactions occur at low antioxidant concentration in a very narrow rang.
Moreover, some phenolic components of essential oils are oxidized by contact with ROS ,consequently producing very reactive phenoxyl radical which add to ROS released by mitochondria and these type of radical reactions enhanced by the presence of cell trasition metal ions such as Fe+2 ,Cu+2,Zn+2and Mg+2 [Stadler et al .,1995 andSakihama et al .,2002].
In addition, our results demonstrated that AEC has anti-brotic effect, as seen in Table (5) and Fig (1). This could be attributed to diminishing the oxidative stress, which occurred through difenoconazole-treatment. Thus, AEC supplementation lead to reduce inhibitors of metalo protinease [which are a group of zinc-dependant endopeptidases] in tissues ,consequently produce an increase in cellagenase level which degraded of brous tissue . Lin et al .(2003) reported that Cinnamon cassia powder reduced signi cantly the expression of alpha-smooth muscle actin (α -SMA),which play an important role in brogensis and connective tissue growth factor (CTGF), Transforming growth factor beta-1 ,and tissue inhibitor of metallo Proteinse-1 ,which elevated by oxidative stress in Sprague-Dawly rats with acute liver injury -induced by dimethyl-nitrosamine.

Conclusions
In conclusion, the present results suggest that the deleterious effects of difenoconazole in hepatic tissue could be due to the induction of oxidative stress as the results of generation of excessive oxy-radicals .Therefore, AEC to be useful in the attenuation of difenoconazole-induced lipid peroxidation (LPO) and showed more or maximum protective effects of the AEC when administrated once daily at 200mg/ kg BW for 28 days ,where it largely minimized the histopathological changes in hepatocytes associated with difenoconazole toxicity . Consent to publish: All the author consent to publish this research article.

Declarations
Consent to participate: All the author have been participate in this research article.