4.6. Animals Experimental

The animals were acclimatized to the laboratory conditions for approximately seven days before beginning the experiments. The rats were randomized into six groups and each group contained 30 animals, blank group (normal animals), control group (DEN induced group), OB high group (OBH, 24 mg/kg body weight/day), OB middle group (OBM, 12 mg/kg body weight/day), OB low group (OBL, 4 mg/kg body weight/day) and positive group (Positive, 10 mg/kg body weight/day cyclophosphamide). All five groups except the blank group were treated with DEN at 70 mg/kg body weight/week of intragastric for 16 weeks. Oroxin B and cyclophosphamide treatment were started at 6 weeks after the hepatocarcinogenesis induction and continued for 16 weeks. All rats were sacrificed after 16 weeks [32,33,34].

The 8–10 surviving rats in each group were sacrificed under anesthesia induced with 3% pentobarbital sodium. Serum was obtained from the blood by centrifugation at 3000 rpm for 15 min at 4 °C, and stored at −80 °C for ELISA analysis. The livers were resected after the rats were sacrificed, then the left lobe of each liver were fixed in 4% paraformaldehyde solution for histopathological examinations. The remaining livers were stored at −80 °C for RT-PCR and Western blot analysis.