3.3.2. Sirius Red and Masson's Trichrome Staining

Sirius Red and Masson's trichrome staining of hepatic cells are shown in Figures 2 and 3.

**Figure 2.** Effect of FLE and DLE on hepatic connective tissue fibrosis caused by CCl4 treatment: assessment by Masson's trichrome stain (G × 200). (**A**) Control, (**B**) CCl4-treated, blue-stained zone indicates increasing fibrosis. (**C**) FLE-treated (30 mg/kg), and (**D**) co-treated with CCl4 and FLE (30 mg/kg); (**E**) DLE-treated (30 mg/kg) showing normal structure and (**F**) co-treated with CCl4 and DLE (30 mg/kg) showing results close to the control.

Microscopic observations of the hepatic tissues of control group (Figure 2A) and those of groups pretreated with FLE and DLE (Figures 2 and 3C,E) show a normal distribution of collagen fibers between the portal spaces and the centrilobular vein manifested by a low red and blue color. After treatment with CCl4 (Figures 2 and 3B), fibrous collagen bridges (shown by the intense red and blue stains) were formed between portal spaces and the centrilobular, marking hepatic fibrosis.

**Figure 3.** Micrograph showing the effect of FLE and DLE on hepatic fibrosis caused by CCl4. Assessment by Sirius red stain (G × 200). (**A**) Control, (**B**) CCl4-treated, showing intensified red stain. (**C**) FLE-treated (30 mg/kg), and (**D**) co-treated with CCl4 and FLE (30 mg/kg). (**E**) DLE-treated (30 mg/kg) showing normal collagen deposition and (**F**) co-treated with CCl4 and DLE (30 mg/kg).

Concerning the association with FLE and DLE, the study revealed that these extracts exerted a protective effect against CCl4-induced hepatic fibrotic scarring, observed through the decreasing red and blue staining when compared to that of control (Figures 2 and 3D,F).

#### *3.4. E*ff*ects of Olive Leaf Extracts on the DNA Fragmentation*

As shown in Figure 4, the DNA extracted from the hepatic sections has changed qualitatively. The control group DNA (lane 1) and that of FLE or DLE-treated groups (lanes 3 and 4) showed an intact band. However, we observed that DNA degraded after treatment with the CCl4 (lane 2 and lane 6), marked by an obvious smearing and laddering which indicates apoptosis. In the FLE + CCl4 and DLE+ CCl4 rats (lane 5 and lane 7), the olive leaf extract addition shows its effect on preventing DNA damage and provides results close to those of the control rats, as observed in Figure 4.

**Figure 4.** Agarose gel electrophoresis of DNA fragmentation. Lane 1: control hepatic tissue DNA; lane 2 and lane 6: CCl4 intoxicated liver DNA, lane 3 and lane 4: FLE- or DLE-treated liver DNA; lane 5 and lane 7: FLE + CCl4- or DLE + CCl4- treated liver DNA.

#### **4. Discussion**

Despite recent therapeutic advances, many liver diseases remain relentlessly progressive because specific therapies to target the underlying etiologies of the liver diseases are not available. Therefore, the demand for liver transplantation is likely to increase unless more effective therapeutic and anti-fibrotic agents are developed [45].

In this research work, healthy adult male albino rats were used as an experimental model for the evaluation of olive leaf extracts on induced hepatotoxicity. Among the known hepatotoxic agents, CCl4 is the best model of oxidative stress-induced liver damage and is commonly used to assay the hepatoprotective capacity of drugs [46].

The obtained results revealed that the oral gavage of SC-CO2 olive leaf extracts effectively attenuated the CCl4-induced hepatic damage and fibrotic scarring in rats. The hepatotoxin-caused injury was marked through the antioxidant enzyme markers' determination in the study of hepatic tissues and DNA. The results obtained in our current study are in accordance with those reported in previous works related to the impact of CCl4 on the liver [39,46–51].

CCl4 is reported to produce free radicals, which affect the cellular permeability of hepatocytes, subsequently leading to elevated levels of serum biochemical parameters such as ALT, AST and ALP [51]. This marks a damaged structural and functional integrity of the liver cell membranes, since these cytosolic enzymes are only released into circulation after hepatic cellular damage. An increase in ALT enzyme activity is almost always due to hepatocellular damage and is usually accompanied by an increase in AST and ALP. Oral administration of both fresh and dried olive leaf extracts produced a significant decrease in serum ALT, AST and ALP levels in CCl4-induced rats, pointing to their hepatoprotective effect. The reduced concentrations of these markers as a result of olive leaf extract administration observed during the present study might be due to the presence of bioactive compounds such as phenolic and triterpenoid compounds; although, there is a difference observed between fresh and dried leaf extracts (Table 1). As in the present study, previous research works on olive leaf extracts showed that CCl4 caused significant elevation of ALT, AST, ALP activities, while pretreatment with

olive leaf extracts significantly suppresses the increase in their levels [24,25]. It has been reported that effective hepatoprotective agents must suppress the increase in ALT activity and bring it closer to a normal level in order to induce liver-healing. This would suggest that agents that can lower ALP levels may be useful in hepatoprotection [9].

The fluidity of the biological membrane as well as some enzyme activities can be affected by lipid peroxidation [36]. In our research work, we observed how treatment with FLE and DLE extracts significantly reduced lipid peroxidation level in CCl4 treated rats, which suggests that the lipid-oxidation inhibition was exerted by the SC-CO2 olive leaf extracts. The generated toxic radicals could be masked through the antioxidants present in the extracts. Amongst the reactive species marker of oxidative stress in cells, we find the highly reactive compound MDA [6]. An increase in MDA levels in tissue sections as a result of CCl4 treatment signifies that lipid oxidation has increased, which subsequently causes tissue injury and the intrinsic cell system failure to eliminate exogenous hepatotoxin agents. In our study, the treatment with SC-CO2 olive leaf extracts showed a significant reduction in lipid peroxidation levels in CCl4-treated rats, demonstrating the capacity of the extracts to reduce such severe alterations. The same behavior was observed for the elevated level of protein carbonyl groups considered as an indicator of protein oxidation. Our results are in agreement with those reported in the literature. In fact, when investigating the protective activity of dried olive leaf extract on CCl4-induced liver damage in rats, the researchers reported that the concentration of MDA significantly increased in rats administered CCl4 when compared to the control group [25]. A similar observation for increasing MDA levels was reported in another study [24] after which a significant decrease was registered in pretreated rats with (methanolic or butanolic) olive leaf extracts in comparison to CCl4-treated rats. Moreover, ethanolic olive leaf extract produced a significant decrease in lipid peroxidation levels in the liver of rats with fluoxetine-induced hepatotoxicity [30].

GPx is an enzyme with peroxidase activity which assures the protection from oxidative damage [9]. GPx catalyzes the reduction in H2O2 or organic peroxide to water or alcohol [52]. Other important antioxidant enzymes are SOD and CAT. SOD is a key enzyme with the ability to convert superoxide radicals into hydrogen peroxide and molecular oxygen, while CAT enhances the conversion of H2O2 to water and molecular oxygen.

In this study, high activities of GPx, SOD and CAT were observed following CCl4 intoxication. Similar observation for SOD was reported in another research on the hepatoprotective effect of extracts of *Cnestis ferruginea* against CCl4-induced toxicity [46]. High antioxidant enzyme activities of tissues and organs do not mean efficient protective potential against oxidative stress caused by CCl4 intoxication. It was found that antioxidant enzyme mRNA expression and enzyme activity were induced by the exposure of hepatocytes to hydrogen peroxide for 24 h [53]. Thus, the increase in CAT, SOD, and GPx activities observed in the liver are probably partly due to the elevated hydrogen peroxide content generated during the metabolism of CCl4 in the body, which lead to up-regulation of gene expression of antioxidant enzyme mRNA and consequently induced an increase in their activities in the liver tissue [54]. The elevated catalase and GPX activities in liver tissue may indicate a large increase in hydrogen peroxide; however, we cannot state to what degree the process of hydrogen peroxide removal is effective [55].

Additionally, the pretreatment of CCl4-intoxicated rats with FLE or DLE showed a marked decrease in the activities of antioxidant enzymes compared to the CCl4-only group. The protective effect of the extracts in CCl4-treated rats is manifested by their capacity to maintain the redox balance marked by the levels of antioxidant enzymes that changed to a figure close to that registered for the control group [9].

Histopathological studies also support the biochemical analysis. The liver histology was improved after co-treatment with SC-CO2 extracts in comparison to the CCl4 only-treated group, suggesting the hepatoprotective capacity of tested extracts against tissue fibrosis. Olive leaves exerted a significant protective effect against CCl4-induced liver damage as marked by the improvement of serum oxidative stress biomarkers and the histology of tissue sections.

The results also showed that CCl4 administration could induce hepatic fibrosis detected by Sirius Red and Masson's Trichrome staining, as manifested by excessive collagen deposition in the hepatic tissue. These findings were in line with other studies showing that CCl4 is able to induce liver fibrosis [56]. The fibrosis status could be related to the activation of hepatic stellate cells (HSCs) as a result of elevated levels of a variety of inflammatory cytokines induced by CCl4 intoxication, as reported in previous studies [57].

Administering FLE or DLE along with CCl4 significantly reduced liver fibrosis. These data support the efficacy of FLE or DLE in reducing the hepatocellular toxic effects of CCl4 and in suppressing the activation and proliferation of HSCs. This positive effect could be due to inflammation reduction. These findings are in line with recent studies that indicate that herbal bioactive molecules are effective against fibrosis in hepatic tissues [58,59].

Despite that significant effect observed for both DLE and FLE extracts, the different composition of tested extracts highlights the importance of phenolic compounds from different classes and triterpenoids as bioactive compounds. Concerning chemical differences related to phenolic compounds, it has to be taken into account that the DLE extract was characterized by the presence of high quantities of hydroxytyrosol and oleuropein isomer. Evaluating the hepatoprotective effect of oleuropein purified from olive leaves, it was found that its administration during ethanol-induced toxicity in rats improves the antioxidant defense system and reduces the levels of lipid peroxides [5]. In another study, oleuropein- and hydroxytyrosol-rich olive leaf extracts revealed hepatoprotective effects against high-fat diet-induced metabolic disorders in rats [14]. With regard to triterpenoids, maslinic acid was only detected and quantified in the FLE extract. Nevertheless, the hepatoprotective effects of triterpenoids present in SC-CO2 olive leaf extracts have not been reported previously.

Since active phenolics and triterpenoids may have not only additive but also synergic effects on hepatoprotection, future investigations are warranted to develop purified triterpenoid extracts and determine correlations between the olive leaf isolated compounds and the hepatoprotective activity parameters.

#### **5. Conclusions**

The findings of the present study revealed the capacity of olive leaf extracts obtained by supercritical CO2 extraction in repairing injuries caused by CCl4-induced hepatotoxicity in a rat model. The obtained results show that fresh and dried olive leaf extracts are able to ameliorate the perturbed biochemical parameters caused by CCl4 treatment. Moreover, SC-CO2 olive leaf extract administration resulted in a reduction in elevated levels of liver lipid peroxidation, and a reduction in protein carbonyls due to CCl4 administration was observed. Serum biochemical markers affirmed the capacity of SC-CO2 extracts to protect DNA, and histopathological studies confirmed the capacity of olive leaf extracts to improve liver fibrosis caused by CCl4 treatment. Therefore, olive leaf contributes to health benefits and it is a potential source of powerful antioxidant compounds. In conclusion, the current research indicates that SC-CO2 extracts from olive leaves have an in vivo protective effect against CCl4-induced injury in rats' livers. Such a biological study is extremely important to highlight the usefulness of olive tree leaves among plants stated as having hepatoprotective activities. Olive leaves, precious by-products from which we have recovered bioactive compounds by green extraction technology, can be reused for agronomic, food, nutraceutical, and pharmaceutical applications.

**Author Contributions:** Conceptualization, A.T., A.F. and J.L.-S.; methodology, A.F. and A.T.; software, A.F., A.E.M.; A.T.; validation, D.A.-R., M.S.A. and L.G.; investigation, A.T.; A.F., A.E.M.; data curation, A.T.; A.F.; writing—original draft preparation, A.T.; writing—review and editing, A.F. and J.L.-S.; visualization, D.A.-R.; supervision, R.M. and D.A.-R.; project administration, R.M., A.S.-C. and J.L-S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Tunisian Ministry of Higher Education and Scientific Research (LR15CBBC05), the Spanish Ministry of Economy and Competitiveness (MINECO) (project AGL2015-67995-C3-2) and Junta de Andalucia, Andalucian Government, Spain (B-AGR-466-UGR18).

**Acknowledgments:** The authors would like to thank Rached Raddadi, Hafedh Trabelsi, and Jihen Jeffel, technicians at Anatomopathology Laboory, Gafsa, Tunisia, for their assistance in histological studies.

**Conflicts of Interest:** The authors declare no conflict of interest.
