Antagonist GR127935 and ZJW Preparation

The 5-HTR1D antagonist GR127935 (GR) (TOCRIS, Ellisville, USA) was dissolved in phosphate buffer saline (PBS) and then diluted into different concentrations. ZJW was prepared using a formula of Chinese herb Rhizoma Coptidis (60 g) and Evodia rutaecarpa (10 g), at a ratio of 6:1. All the herbs were purchased from Longhua Hospital herbal pharmacy department. Briefly, the mixture (70 g) was extracted twice for 1 h each time by refluxing in ethanol (1: 8, v/v). The filtrates were concentrated and dried in vacuum at 60 °C. Its preparations were standardized, regulated, and quality controlled according to the guidelines defined by China Food and Drug Administration (CFDA). High-performance liquid chromatography (HPLC) was used to identify the components of ZJW extract, and confirm the final concentration of ZJW extract to ensure the quality and stability. Detailed procedures were followed with the published protocol [6 (link)]. ZJW was dissolved in PBS and diluted into three different concentrations, 25, 50 and 100 μg/mL, corresponding to low, medium and high dose of ZJW (ZJW-L, ZJW-M and ZJW-H).