Experimental design

The animals (n = 28) were randomly divided into four groups (n = 7/group). Group 1 was considered as a negative control group. In group 2 [Ehrlich ascites carcinoma (EAC) solid tumor (EST) group] mice were intramuscularly injected with a single dose of 0.2 mL EAC (2 × 10⁶ cells) in the femoral region at day zero (D0) [37] and by vehicle (DMSO-PBS) at D12 (the day at which the tumor was developed). In group 3 (low dose-treated group) mice were intratumorally injected with A. machrostachyum methanolic extract (AME, at a dose equals 1/10 LD_50; 180 mg/kg body weight) at D12 trice weekly for 2 weeks. In group 4 (high dose-treated group) mice were treated as in group 3 but with a dose equals to 1/5 LD_50; 360 mg/kg body weight.

At the end of the experiment (D25), mice were euthanized (by decapitation under ether anesthesia) and the tumors were weighed and divided into the following 3 parts: the first part was preserved in 10% formalin (for histopathology), the second part was kept in − 80 °C (for real-time PCR and western blot), and the third part was frozen at − 20 °C (for Elisa and comet assay).