Aucubin

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.

Fresh bone marrow-derived mesenchymal stem cells (BM-MSCs) were isolated from 4-week-old C57BL/6 mice according to previous publication [24 (link)]. Briefly, bone marrow was flushed out with a syringe and needle, and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were incubated at 37°C with 5% CO₂ for 7–10 days until nonadherent cells were removed by changing the medium. RAW264.7 were cultured in DMEM (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Carlsbad, CA) at 37°C with a humidity of 5% CO₂. Aucubin was purchased from Yuanye Bio-Technology (Shanghai, China), dissolved in DMSO, and stored at −20°C for usage.