In vivo anti-tumor evaluation
All animal experiments were approved by the Institutional Animal Care and Treatment Committee of Chengdu University of TCM (Chengdu, P. R. China). Female Balb/c nude mice and Balb/c mice (6-8 weeks old) were purchased from HFK Bioscience CO., Ltd. (Beijing, China). These Balb/c mice or Balb/c nude mice were inoculated with 100 μL of 4T1 cell suspension (1.0×106cells) or MDA-MB-231 cell suspension (1.0×107 cells). At 5 days after inoculation, when the tumor volume had reached about 100 mm3, the mice were equally and randomly divided into three treatment groups (control, 20 mg/kg, and 40 mg/kg) (n=6). Mice received icariin treatment by intraperitoneal injection once daily for two weeks. The tumor volume (0.5 × L × W2, where L is the length and W is the width) and body weight of the mice were recorded every two days. The mice were euthanized by cervical dislocation at the termination of the experiments. The tumors were isolated, imaged, weighted, and fixed with paraformaldehyde for further immunohistochemistry evaluation.