## 2 Materials and Methods
### In Vivo Hepatoprotective Activity against Paracetamol-Intoxicated Wister Albino Rats
#### 2.9.2 Assessment of In Vivo Antiradical Potential of Liver Hepatic Tissues  
Assessment of liver hepatic tissues for in vivo antioxidant level was performed by following [PERSON] et al.'s [42] method. Before liver excision, the serum blood samples were collected via tail vein using sterile centrifuge tubes containing heparin from rats in the individual groups. Then, the rats in all groups were humanly sacrificed to excise the liver. The excised livers were made free of blood traces by rising with ice-cold normal saline. Later, the weight of the livers was noted, followed by homogenization of individual liver tissues in 0.1 M cold phosphate buffer (pH 7.4) to obtain liver hepatic tissue homogenate. Subsequently, liver tissue homogenates post-mitochondrial supernatant (PMS) were obtained with the application of centrifugal force at 4 \({}^{\circ}\)C maintained up to 10 min at 10,000 rpm. The tissue homogenate was re-forced with centrifugal force for an hour at 17,000 rpm. Finally, liver tissue homogenates were taken for assessment using the liver function test, which was achieved by determining the reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), antioxidant, and lipid peroxidation (LPO) levels.  
Measurement of Glutathione  
The reduced glutathione analysis was completed with the use of the [PERSON] et al. [43] procedure, including minor changes. In this experiment, homogenized liver tissues (1 mL) were reacted with the 6 mL phosphate buffer solution--PBS (potassium hydrogen phosphate--9400 \(\mu\)L and potassium di-hydrogen phosphate--3000 \(\mu\)L were mixed) at a pH of 8 and 1000 \(\mu\)L of 0.6 mM 5,5\({}^{\prime}\)-dithiobis (2-nitrobenzoic acid)-DTNB (2.38 mg DTNB was dissolved in 10,000 \(\mu\)L methanol), followed by further incubation for 600 s at room temperature. The absorbances were taken at 412 nm. Glutathione content was measured by plotting the graph of standard, which was examined with the application of a similar experimental procedure and expressed as the number of moles per milligram of protein (nmol/mg of protein).  
Measurement of Catalase  
Catalase concentration estimation was obtained using the Claiborne method [44] with a few alterations. During the analysis, 50 units of homogenized liver tissue were treated with 1950 \(\mu\)L of phosphate-buffered saline (PBS--50 mM) at a pH of 7 and 1 mL of 0.7 mM hydrogen peroxide--H\({}_{2}\)O\({}_{2}\) (0.160 mL of H\({}_{2}\)O\({}_{2}\) added to 100 mL PBS). The record absorbances were noted at 240 nm for 0 min and 1 min, which were measured as nmol/mg of protein.  
Measurement of Superoxide dismutase  
The superoxide dismutase level was assessed using the [PERSON] et al. [45] method, with some alterations. A solution of sodium carbonate buffer, hydrochloric acid (0.5), and epinephrine was prepared for the analysis. To the homogenized liver tissue (0.1 mL), the sodium carbonate buffer (0.8 g) with epinephrine solution was added and taken into the cuvette. The second cuvettes with sodium carbonate buffer (0.8 g) and homogenized liver tissue (0.1 mL) were combined and analyzed at 295 nm with an auto-analyzer (CPC StatFax 3000 Plus, Shimadzu, Kyoto, Japan) for 0 min and 1 min and defined as nmol/mg of protein.  
Measurement of Lipid Peroxidation  
With some modifications, LPO activity was measured using the method of [PERSON] et al. [42]. The homogenized liver tissue was assessed to identify the concentration of thiobarbituric acid. During the experiment, 10% tissue homogenate (0.5 mL) was treated with 15% trichloroacetic acid--TCA (0.3 mL) and 0.375% thiobarbituric acid--TBA (0.3 mL) along with 5 N HCl (0.03 mL) followed by a 15 min incubation at 95 \({}^{\circ}\)C in a hot water bath. Later, the solutions were brought to normal temperature and subjected to a centrifugal force of 2000 rpm. Thereafter, the resultant separated solution was taken for analysis, which was read for absorbance at 535 nm in contrast to the suitable blank. The lipid peroxidation reaction was calculated for nmol/mg of protein.