In our work, we evaluated the hepatoprotective activity of EOE by investigating its preventive and antioxidant potentials against CCl4-induced oxidative hepatic injury in rats. Therefore, elevated amounts of biochemical markers like ALT, AST, ALP and bilirubin in addition to the significant raise in total cholesterol and triglycerides indicate cellular membrane impairment in the CCl4 intoxicated rats. Our results demonstrated that polyphenols rich olive leaves extract (EOE) (400 mg/Kg) improved the elevated liver markers quantity close to normal indicating a promising hepatoprotective capacity of EOE.
The raise in ALP activity and total bilirubin level was depleted by EOE treatment. These results are likely to be related to the capacity of the extract to maintain biliary impairment in rats with CCl4-induced liver damage. Many authors, have explained the protective capacity of plants extracts against hepatic injury by their antioxidant potential (Hfaiedh et al., 2014). ROS-induced oxidative stress has a pivotal role in hepatic injury. It could provoke liver damage by triggering lipid peroxidation, affecting biomembrane structure, and altering the enzyme function (Yang et al., 2017).
To overwhelm the damaging actions of free radicals, organisms established enzymatic and nonenzymatic antioxidative systems. Enzymatic systems consist essentially of SOD, CAT, and GSH-Px that protect tissues from oxidative injury through radical scavenging mechanism. Thus, these antioxidant enzymes play a vital role in hepatic detoxification (Huo et al., 2011). Superoxide dismutase converts superoxide radical (•O2) into hydrogen peroxide (H2O2) and oxygen (Urrutia-Hernández et al., 2019). Catalase decomposes hydrogen peroxide to generate water and oxygen. Glutathione peroxidase catalyzes the reduction of
In this investigation, CCl4 intoxication led to a significant reduction in the amounts of antioxidant enzymes (SOD, CAT, and GSH-Px), demonstrating drastic oxidative stress status in hepatocytes (Chen et al., 2016). Pretreatment with EOE at 400 mg/kg resulted in restoration of the activities of SOD, CAT and GSH-Px comparable to silymarin. These results reflect those of Ustuner et al. (2018) who also found that O. europaea leaf regulated the antioxidant enzymes activities of CCl4- intoxicated rats, thus affirm its antioxidant potential. A non-enzymatic system, glutathione, is implicated in cellular protection against oxidative stress-induced damages. Reduced glutathione (GSH) gives an electron to ROS transforming them into non-toxic compounds (Raj et al., 2014).
The results of this study suggest that CCl4 significantly decreased the reduced glutathione (GSH) level. Nevertheless, treatment with EOE (400 mg/Kg) has reestablished the GSH near normal levels. CCl4 intoxication-induced free radicals accelerate lipid peroxidation by removing a hydrogen atom from the unsaturated fatty acids (Jain et al., 2008). Malondialdehyde is the end product of this mechanism, and the increase of MDA in liver tissue is an index of hepatic damage (Yang et al., 2017). One of the main antioxidative mechanisms to stop the lipid peroxidation chain reaction is free radicals scavenging (Jain et al., 2008).
The current study revealed a significant increase in the MDA level in response to CCl4 treatment. O. europaea extract (400 mg/kg) brought the MDA formed near normal levels. According to these data, we can infer that the hepatoprotective capacity of O. europaea leaves could be attributed to their antioxidant and free radical scavenger ability by inhibiting the binding of free radicals to hepatocyte membrane and thus preventing lipid peroxidation (Jain et al., 2008). Several investigations have reported that the hepatoprotective activity could be relevant to the antioxidant system responsible for the ROS scavenging and the prevention of free radicals such as hydroxyl, alkyl and lipid peroxides (Hsu et al., 2009).
In this study, EOE was found to exhibit marked radical scavenging and metal chelating activity, thus, it could reverse pathological impairment created by the CCl4-generated free radicals. Earlier reports disclosed that olive leaves possess potent antioxidant power (Ferreira et al., 2007). It seems possible that the protective effect of EOE is due to the active constituents which were identified by HPLC-MS and previously described to arise in O. europaea L. leaves (Benavente-Garcı́a et al., 2000 ; Meirinhos et al., 2005 ; Pereira et al., 2007). Many phyochemical constituents, including polyphenols, have antioxidant potential, which is associated with the protection of biological system from damaging oxidation reactions (Guex et al., 2018).
The phytochemical screening of O. europaea leaves extract showed that it has high contents of total phenolics, flavonoids and tannins. These results reflect those of Guex et al. (2018) who also found that O. europaea leaves contain a considerable quantity of polyphenols and flavonoids, besides other active compounds. It was reported that phenolic compounds own antioxidant properties considering their structures, which contain hydroxyl groups with mobile hydrogen. These compounds prevent dissociation of hydroperoxides into free radicals which improve the antioxidant capacity of plants extracts (Athmouni et al., 2017).
The results of the current study displayed the protective and antioxidant potential of polyphenols rich ethanol extract from olive leaves against CCl4-induced liver injury in rats. Raj et al. (2014) have noted that the protective activity of polyphenols from Amorphophallus commutatus var. wayanadensis againt CCl4-induced hepatic toxicity is liable to their antioxidant activity. Phenolic profiling of EOE revealed the presence of hydroxytyrosol, tyrosol, vanillin, verbascoside, oleuropein, apigenin-7-O-rutinoside, luteolin-7-O-glucoside, luteolin and apigenin.
The significant hepatoprotective and antioxidant capability of EOE could be attributed to its polyphenolic composition. This study supports evidence from another study which was performed to explore the therapeutic action of an olive leaves extract containing twenty percent oleuropein on CCl4-induced hepatic injury (Ustuner et al., 2018). They suggested that olive leaf extract reduced hepatic toxicity by lowering MDA levels, regulating antioxidant enzymes activity, and decreasing DNA impairment. In addition, olive leaf extract decreased ALP, AST, and ALT levels, and raised SOD and CAT activities of blood samples.
In accordance with the present results, previous studies have demonstrated that administration of oleuropein and hydroxytyrosol olive leaf extracts (16 mg per kg b.w.) restored the raised amounts of TG and hepatic enzymes through improving SOD and CAT activities. In addition they reported that the olive leaves extracts reduced the expression of NF-κB and TNF-α (Mahmoudi et al., 2018). Pan et al. (2013) reported that hydroxytyrosol at 1 and 10 mg/kg attenuated ischemia/reperfusion-induced hepatic damage, demonstrated by the reduction of AST and ALT levels, reduction of histopathological changes, and inhibition of cell apoptosis. They attributed the protective capacity of hydroxytyrosol to its anti-inflammatory and antioxidative activity.
A previous study had noted that hydroxytyrosol (0.5 mg/kg; oral), and tyrosol (30 mg/kg; i.p) reduced TCDD-induced liver injury by suppressing CYP1A1 expression, and boosting the liver antioxidant enzymes. These compounds suppressed apoptosis through the inhibition of Bax expression, and the induction of Bcl-2 expression (Kalaiselvan et al., 2015). Makni et al. (2011) revealed in their study that vanillin (150 mg/kg) inhibited the decrease of protein synthesis and the augmentation in plasma ALT and AST and attenuated TNF-α, IL-1β, and IL-6 expression levels. They also revealed in their study that this compound prevented liver lipid peroxidation and formation of protein carbonyl and preserved the antioxidative systems.
Zhao et al. (2009) assessed the hepatoprotective potential of acteoside (50, 150 and 300 mg/kg) on Bacillus Calmette-Guerin plus lipopolysaccharide-caused immunological hepatic damage. They concluded that acteoside decreased the raised hepatic index, hepatic AST and ALT contents, liver NO and MDA levels, and re established liver superoxide dismutase action. The expression of Bax was reduced, while the expression of Bcl-2 was elevated. Another significant finding was that oleuropein (200 mg/kg) inhibited the stimulation of hepatic stellate cells caused by the TNF-β1, besides the activation of caspase-3.
These results were likely to be related to the induction of heme oxygenase-1 by the NF-E2-related factor 2 (Domitrović et al., 2012). Kim et al. (2010) explained the molecular process for the hepatoprotective action of oleuropein by the blockage of the expressions of the liver fatty acids related genes. Further, they revealed in their investigation that oleuropein decreased the expression of the oxidative stress reactions related genes, and the pro-inflammatory genes. Lee et al. (2011) established that luteolin attenuated GalN/LPS-induced apoptosis and nuclear phosphorylated c-Jun levels.
The authors associated this protection to the blockage of the extrinsic and intrinsic apoptotic pathways. Further reports demonstrated that luteolin inhibited the evolution of hepatic fibrosis by suppressing fibrosis-related genes in HSC and inhibiting TGF-β and PDGF signaling pathways (Li et al., 2014). Apigenin (10 mg/kg) was described for its potent antioxidant effect and hepatoprotective potential. As mentioned in the investigation of Rašković et al. (2017), apigenin treated animals exhibited reduced ALT and ALP activity. Paracetamol-induced histopathological alterations were reduced by apigenin. Apigenin also reversed elevation in MDA level. In addition, apigenin increases the enzyme antioxidant defense mechanisms.
The findings of this work demonstrated that the phenolic-rich ethanol extract of olive leaves decreased CCl4-induced liver damage by recovering antioxidant enzymes activities, lowering lipid peroxidation and restoring the levels of liver markers. These data suggested that the hepatoprotective potential of the extract could be attributed to its antioxidative action. However, supplementary analyses are indispensable to ascertain the molecular mechanisms of action of O. europaea L. active components.