In vitro hepatoprotective and apoptotic signaling assays

HepG2 cells were seeded (0.5 × 10⁵ cells/well, in triplicate) in a 96-well flat-bottom plate and grown over night. DCFH (IC₅₀: 100 μg/mL) was used as a chemical inducer of hepatocyte toxicity [9–11]. Four different doses of SSEE (25, 50, 100 and 200 μg/mL) and DCFH were prepared using dimethyl sulfoxide (DMSO; > 0.1%, final) and complete RPMI media. For hepatoprotection assay, the cultures were treated with DCFH (100 μg/mL) plus a dose of SSEE, including untreated and DCFH alone-treated controls. The cells were incubated for 48 h, following MTT assay (TACS MTT Cell Proliferation Assay Kit, Trevigen, USA) as per the manufacturer’s instruction, and the optical density (OD) was measured (Microplate Reader ELx800; BioTek, USA).

For anti-apoptotic assay, caspase-3/7 activation was measured (Apo-ONE-homogenous caspase‑3/7 Assay Kit; Promega, USA) as per the supplied manual. Briefly, HepG2 toxicity was induced with DCFH as above, and treated with the four doses of SSEE for 48 h. Caspase-3/7 reagent was added (100 μL/well), incubated in dark at RT for 5 h, and the OD was measured.

For both the assays, cell survival was determined using the equation: [(OD_s-OD_b)/(OD_c-OD_b)] × 100 where OD_s, OD_b and OD_c are the optical density of sample, blank and negative control, respectively. Data were subjected to non-linear regression analysis (Excel software 10.0, Microsoft, CA, USA) and presented as % cell survival in relation to the untreated control.