## 2 Materials and Methods
### In Vivo Hepatoprotective Activity against Paracetamol-Intoxicated Wister Albino Rats
#### Measurement of Aspartate Transaminase  
AST concentration determination was performed using the procedure of [PERSON] et al. [39] with minor modifications. The fresh working reagent was obtained with the combination of Reagent 1 and Reagent 2 (4:1). Distilled water was used as a blank to form a baseline in the spectrometric readings. In the analysis of the test serum sample, the serum (50 \(\upmu\)L) was treated with a working reagent (500 \(\upmu\)L) at a 37 \({}^{\circ}\)C temperature. The NADH oxidation rate was recorded at 340 nm using an auto-analyzer (CPC Stat Fax 3000 Plus, Shimadzu, Kyoto, Japan) by recording a decrease in absorbances. Finally, the level of AST was measured in terms of units per liter of blood serum (U/L).  
Measurement of Alanine Transaminase  
An ALT level was measured using the reported method of [PERSON] et al. [39] with certain modifications. In the experiment, preparing the working reagent involved mixing Reagent 1 with Reagent 2. Distilled water served as the blank. Then, the test serum was analyzed by reacting 0.05 mL of the test serum with 0.5 mL of a working reagent at a 37 \({}^{\circ}\)C temperature. The decrease in absorbances was noted at 340 nm using an auto-analyzer (CPC Stat Fax 3000 Plus, Shimadzu, Kyoto, Japan) to measure the NADH oxidation rate. The level of ALT was measured in U/L.  
Measurement of Alkaline Phosphatase  
An ALP concentration assessment was performed using the [PERSON] et al. [40] protocol, including a few variations. During analysis, test serum (20 \(\upmu\)L) was mixed with 1 mL of ALP reagent (Reagent 1). The yellow color that formed was kinetically measured, which caused changes in absorbance, recorded at 405 nm with an auto-analyzer (CPC Stat Fax 3000 Plus, Shimadzu, Kyoto, Japan). Changes in absorbances are proportional to the ALP level of the test serum. ALP amount was expressed in U/L.  
Measurement of Total Bilirubin  
The total bilirubin level estimation was performed following the Diazo method [41]. During the experiment, a fresh working reagent was made by mixing Reagent 1 (total bilirubin reagent) and Reagent 2 (sodium nitrite reagent). For the blank, distilled water (25 \(\upmu\)L) was combined with a working reagent (500 \(\upmu\)L). Similarly, the test serum (25 \(\upmu\)L) was reacted with a working reagent (500 \(\upmu\)L) in the test serum sample analysis. Then, the solutions were homogenized well, followed by 5 min of incubation (37 \({}^{\circ}\)C). An auto analyzer (CPC Stat Fax 3000 Plus, Shimadzu, Kyoto, Japan) was used to record the absorbances at 546 nm. Subsequently, the concentration of total bilirubin was expressed as a milligram per deciliter of blood serum (mg/dL).  
Measurement of Cholesterol  
Cholesterol estimation was carried out using the dictated experiment of [PERSON] et al. [39] with minor variations. The polyethylene glycol-cholesterol oxidase-peroxidase-4-aminoantipyrine procedure and lipid clearing factor were implemented for end-point determination. The analysis included the preparation of a standard reagent (solution of 0.1 mL Reagent 1 and 0.01 mL Reagent 2), which contained cholesterol, preservatives, and stabilizers. The blank reference selected was distilled water. The serum sample (10 \(\mu\)L) of rats was reacted with the methanolic extract of _C. simplicifolia_ leaf combined with an enzyme reagent (1000 \(\mu\)L) followed by a 10 min incubation at 37 \({}^{\circ}\)C. Absorbances of the sample were noted for 505 nm with an auto-analyzer (CPC Stat Fax 3000 Plus, Shimadzu, Kyoto, Japan). Cholesterol was measured as mg/dL.