*3.1. Identification of Phenolic and Triterpenoid Composition in SC-CO2 Olive Leaf Extracts*

In Table 1 are summarized the different compounds detected in both FLE and DLE. The analysis of the extract composition permitted the chemical characterization of 16 compounds. Quantitative results showed in this table include the amount of each identified compound in the administered dose to evaluate the hepatoprotective effects.


**Table 1.** Identified and quantified phytochemicals in the SC-CO2 olive leaf extracts.

Value = X ± SD (μg of compound per 30 mg of extract), not detected (-).

A clear qualitative and quantitative difference was observed between fresh and dried leaves. In fresh leaves only vanillin, lignans (pinoresinol and acetoxypinoresinol), diosmetin and triterpenoids (maslinic, ursolic and oleanolic acids) were detected. This could be related to the impact of drying on the quality and the composition of olive leaf extract. Regarding the administered dose, acetoxypinoresinol was the major phenolic compound and constituted 37 μg in FLE, whereas in DLE, oleuropein formed the major phenolic compound (42 μg) followed by hydroxytyrosol (19 μg). Regarding triterpenoids, ursolic acid was the major compound in both extracts, being higher in fresh olive leaf extract.

#### *3.2. Biochemical Measurements*

#### 3.2.1. Effect on Plasma Biochemical Markers

Table 2 summarizes the impact of the hepatotoxin and olive leaf extract administration on liver function tests. Compared to the control group, the ALT, AST, ALP and LDH activities in serum of rats treated with CCl4 increased significantly (*p* < 0.001) after eight weeks reaching 336, 417, 231 and 261 U/L, respectively, indicating acute hepatocellular damage (Table 2). Nevertheless, the oral administration of olive leaf extracts (dose of 30 mg/kg b.w.) during intoxication showed a significant decrease in AST, ALT, ALP and LDH activities compared to CCl4 only-treated rats.


**Table 2.** Effect of CCl4, FLE and DLE administration on hepatic biochemical markers.

AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline, phosphatase and LDH: lactate dehydrogenase. Values are expressed as mean ± SD of six rats in each group. CCl4 group versus control group: \*\* *p* < 0.01; \*\*\* *p* < 0.001. CCl4 + FLE or CCl4 + DLE group versus CCl4 group: <sup>+</sup> *p* < 0.05; ++ *p* < 0.01; +++ *p* < 0.001.

The observed decrease was more pronounced in terms of AST activity when DLE was administered (389 U/L, *p* < 0.001) during CCl4 toxicity. Contrastingly, FLE administration in intoxicated rats caused a slightly more important decrease than with DLE administration in terms of ALT, ALP and LDH activities (Table 1).

3.2.2. Lipid Peroxidation and Protein Carbonyls

The effect on lipid peroxidation and protein carbonyls contents are summarized in Table 3. For CCl4-intoxicated rats, the TBARS level increased significantly (4.6 nmol MDA equivalents/g tissue) when compared with the control group (1.13 nmol MDA equivalents/g of tissue). Nevertheless, treatment with olive leaf extracts significantly reduced the lipid peroxidation level in CCl4-treated rats to 2.9 nmol MDA equivalents/g tissue in the case of FLE + CCl4 and 2.7 nmol MDA equivalents/g of tissue in the case of DLE + CCl4.

Likewise, protein carbonyl amounts significantly increased (\*\*\* *p* < 0.001) further CCl4 intoxication (2.7 nmol/mg protein) in comparison to the control group (1 nmol/mg protein). However, FLE pretreatment of intoxicated rats showed a more pronounced effect than DLE pretreatment when compared to intoxicated rats (1.6 nmol/mg protein) with values of 1.0, 2.7 and 2.03 nmol/mg protein for control, CCl4 and DLE+ CCl4 treatment, respectively.

A significant increase (\*\* *p* < 0.01) was registered in terms of SOD, CAT and GPx activities among those in the CCl4-treated group (141 U/mg protein, 136 μmol of H2O2 destroyed/min per mg protein and 100 nmol of NADPH oxidized/min per mg protein, respectively) when compared to the control group (89 U/mg protein, 109 μmol of H2O2 destroyed/min per mg protein and 83 nmol of NADPH oxidized/min per mg protein, respectively). In contrast, SOD, CAT and GPx activities significantly decreased (*p* < 0.05) in intoxicated rats pretreated with DLE or FLE when compared to the rats who received only CCl4 (Table 3).



Values are expressed as mean ± SD of six rats in each group. CCl4 group versus control group: \*\* *p* < 0.01; \*\*\* *p* < 0.001. CCl4 + FLE or CCl4 + DLE group versus CCl4 group: + *p* < 0.05; ++ *p*<0.01; +++ *p*<0.001.
