YCHT Herbal Extract Preparation and Analysis

YCHT consists of Artemisia annua L. (Shanxi, China), Gardenia jasminoides Ellis (Jiangxi, China), and Rheum Palmatum L. (Gansu, China) in a 4.5: 1: 1.5 ratio. The medicinal herbs were purchased from Shanghai Huayu Chinese Herbs Co., Ltd. (Shanghai, China) and extracted according to our previous published patent⁶⁰ . Artemisia annua L. (2250 g) was first boiled in 600 L of water for 1 h, and then Gardenia jasminoides Ellis (500 g) and Rheum Palmatum L. (750 g) were added in together. Finally, the filtered solutions were concentrated into the aqueous extracts containing 0.48 g/mL raw herbs. Then the YCHT extract was dissolved in methanol and filtered through a membrane syringe filter (0.2 µm) (Millipore Co., Bedford, MA, USA) and subject to HPLC analysis. An Agilent 1200 HPLC system (Santa Clara, CA, USA) coupled with a binary pump, an automatic injector, and a diode array detector were used in this study. The separation was carried out on a Phenomenex Luna C18 (250 × 4.6 mm I.D., 5 µm) (Phenomenex, Torrance, CA, USA). The column was eluted with mobile phase (A) 0.1% acetic acid in water and (B) methanol using a linear gradient of 5–62% B over 0–75 min, 62–73% B over 75–80 min, 73–100% B over 80–85 min, held at 100% B for 10 min, then equilibration at 5% B. The flow rate was 1 mL/min and the injection volume was 20 µL. All the samples were kept at room temperature during the analysis. The detection wavelength was set at 260 nm. Agilent ChemStation software was used for peak identification and integration and the content of the constituents was calculated using standard curves of gallic acid, chlorogenic acid, geniposide, and crocin.