Geniposidic acid

The aqueous extract was prepared by boiling under optimum conditions, and the filtrate was freeze-dried with a Heto PowerDry LL3000 freeze dryer (Thermo, USA) and stored at −80°C. The components and content of Eucommia leaf extracts were determined by HPLC. The standard chemicals (aucubin, geniposidic acid, carnosine, catechin, isoquercitrin, chlorogenic acid, and astragaloside) were purchased from Shanghai Yuanye Biotechnology Co., Ltd (Yuanye, China). HPLC analysis of ELAE was performed using a 2695 liquid chromatographic system equipped with an inverted C18 column (Waters, USA). The mobile phase was a gradient elution system composed of water containing acetonitrile (A)-0.1% phosphoric acid solution (B), with a flow rate of 1 ml/min, and a column temperature of 35°C. Acetonitrile (A)-0.1% phosphoric acid solution (B) was eluted in a gradient manner (0–7 min, 5% A; 7–9 min, 5% A ⟶ 8% A; 9–28 min, 8% A; 28–30 min, 8% A ⟶ 20% A; 30–42 min, 20% A; 42–43 min, 20% A ⟶ 50% A; 43–63 min, 50% A; 63–64 min, 50% A ⟶ 5% A). A photodiode array (PDA) detector was set at 254 nm, and the online UV spectrum was recorded within the range of 200–300 nm.

Standard compounds geniposide, gardenoside, genipin-1-gentiobioside, shanzhiside, chlorogenicacid, crocin-1, crocin-2, crocetin, geniposidic acid, and genipin with over 98% purity were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Cell Counting Kit-8 purchased from EnoGene technology Co., Ltd. (Nanjing, China). Annexin V-FITC/PI apoptosis detection kit purchased from BestBio technology Co., Ltd. (Shanghai, China). DAPI staining solution purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). TNF-α,IL-6, and NO enzyme-linked immuno sorbent assay (ELISA) kits (R&D, USA) were purchased from Changchun Bestgene Biotechnology Co., Ltd. (Changchun, China). ALT, AST, ALP, MDA, SOD, and GSH kits were obtained from Nanjing JianCheng Institute of Biological Engineering (Nanjing, China). Acetonitrile and Formic acid (Fisher, Fair Lawn, NJ, USA) were of HPLC grade.