In Vitro Hepatoprotective Assay

The in vitro hepatoprotective assay was based on the protection of human liver-derived HepG2 cells against CCl₄-induced damage. We procured HepG2 cell line from American Type Culture Collection no. (ATCC#HB-8065) (American Type Culture Collection (ATCC), Manassas, VA, USA). Human liver hepatocellular carcinoma cells (HepG2) were grown in DMEM media supplemented with 10% bovine serum, 0.1% antimycotic solution from (Sigma-Aldrich Co., St Louis, MO, USA) at 37°C in a humidified chamber with 5% CO₂. The cells were then treated with medium containing 1% CCl₄ along with or without the test compounds. Silymarin was used as the reference drug. Stocks of all compounds (1.0 mg/mL) were made in DMSO and further dilutions (100 µg/mL) were prepared in culture media. Cells were treated with test compounds, separately; with dose of 25 µg/mL of each compound. Treated cells were incubated in complete growth media for 48 hours. As a positive control, cells were also tested with the reference drug, silymarin. Cytotoxicity was assessed by calculating the viability of the HepG2 cells by MTT reduction assay.25 (link)