Barbital sodium

The rats were anesthetized with 2% barbital sodium (2.3 ml/kg; Sigma-Aldrich) prior to hyperthermia treatment. The rats were then placed under the magnetic field and the body temperatures were measured rectally.
Rats in group M were exposed to the magnetic field for 30 min; rats in groups H1 and H2 were placed under the magnetic field to ensure that the major axis of the thermoseeds and the reversal magnetic field direction were parallel. Three electric thermocouples were inserted in order to monitor the temperature at the center of the tumor, at the tumor edge and the body temperature, separately. A fixed electric current (50 Hz) was applied to groups H1 and H2 to heat the thermoseeds (for ~2 min to heat to the desired temperature). The treatment was maintained for 30 min for the H1 group and 10 min for the H2 group, respectively.

Fourteen days after the treatment, four rats were randomly selected from each group and sacrificed by an intrapertoneal injection of barbital sodium (Sigma-Aldrich). The tumors were removed, fixed in 10% formalin and paraffin-embedded sections were prepared. Hematoxylin and eosin staining of the tumor tissue was performed and the pathological changes were visualized under a microscope (Nikon Eclipse Ci-E, Nikon, Beijing, China).

Dry ZSS was obtained from Hebei Huadu Pharmaceutical Co., Ltd (Hebei, China) and GABA was obtained from Shanghai Toong Yeuan Food Tech Co., Ltd (Shanghai, China). Diazepam (Diaz) was purchased from Shandong Xinyi Pharmaceutical Co., Ltd (Shandong, China). Sodium pentobarbital and sodium barbital were obtained from Sigma-Aldrich (St. Louis, MO, USA). All reagents and solvents were commercially available as analytical grade.
Dry ZSS was washed and then pulverized after drying. The powders were screened through 6-mm mesh, distilled water was added, and the mass concentration of crude ZSSG was set at 1.55 g/mL. The mixture was refluxed for 1 h and this process was repeated three times. Subsequently, a high concentration ZSS extract was prepared by a rotary evaporator (vacuum − 0.08 to − 0.10 MPa) at 50–70 °C and then dried under reduced pressure (− 0.08 to − 0.10 MPa, 50–70 °C). After preliminary animal experimental exploration, the dry powder of the ZSS extract and GABA was weighed and mixed in a 63:50 ratio (equivalent to mass ratio of raw ZSS and GABA: 6/1). Finally, the solution was filtered through a 0.22 membrane and the compounds of ZSSG were identified by ultra-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (UPLC-Q/TOF–MS) analysis.