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Cell Culture and Determination of Cytotoxicity. Metabolically  active H4IIE rat hepatoma cells were grown in Dulbecco¡¯s modified  Eagle¡¯s medium (4.5 g/L glucose, 2 mmol/L L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum)in a humidified atmosphere (37 ¡ãC, 5% CO2). The effect of isolated  compounds on cell viability was determined using the MTT assay.
The cells were plated in 96-multiwell plates with 10 000 cells/well.The cells were allowed to attach for 24 h and then treated with different  concentrations of the compounds for 24 h. After this treatment the  medium was changed and the cells were incubated for 3 h under cell culture conditions with 0.7 mg/mL MTT. After this incubation the  cells were lysed with 50% EtOH/49% H2O/1% HOAc. The
concentration of reduced MTT as a marker for cell viability was  measured at 560 nm. Data are given as mean SEM of at least  three independent experiments. The significance of changes in the  test responses was assessed using a one-way ANOVA followed by  LSD test (Analyze-it, Leeds, UK); differences were considered
significant at P 0.05.
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