Preparation of Indigo Naturalis and High Performance Liquid Chromatography (HPLC) Analysis

Indigo naturalis concentrated granules were purchased from the Pharmacy Department of Dongfang Hospital, Beijing University of Chinese Medicine (Beijing, China). The herb was extracted using a method of stimulated family decoction, then the extracts were concentrated, dried to form granules. This process was performed by Beijing Tcmages Pharmaceutical Co., Ltd. (Beijing, China) according to Good Manufacturing Practice (GMP) for Drugs (Chinese FDA) to guarantee the quality control. HPLC was carried out to identify the main chemical constituents. The reference standards of indigo and indirubin were purchased from National Institutes for Food and Drug Control (Beijing, China). Then, these two major components of indigo naturalis were analyzed by Agilent 1260 series HPLC system (Agilent Technologies, Palo Alto, CA, United States). The chromatographic separation was performed with Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 μm) column (Nouryon, Bohus, Sweden) under isocratic flow of 55%–80% acetonitrile in 0–15 min at 25°C (1.0 mL/min). The injection volume was 10 μl and the wavelength of the detector was set at 286 nm.