Bilobalide

Obtaining rabbit PB and BM and from them isolating mononuclear cells (MNCs) refer to our previous report.4 Human PB MNCs were isolated in the same way. Rat BM was harvested from the femurs and tibias of male Sprague‐Dawley rats (250‐280 g, supplied by the Laboratory Animal Center of Xi'an Jiaotong University) killed with inhalation of CO₂. MNCs were plated on 100‐mm culture dishes that had been coated with human fibronectin (for PB OECs) or rat collagen type I (for BM OECs), and were cultured for over 2 weeks in endothelial growth medium‐2 (EGM‐2, Lonza‐BioWhittaker, Walkersville, MD, USA) at 37°C, letting the included OECs increased while other cell types disappeared. The medium was changed every other day. Cells were subcultured when they approached 90% confluence. For bilobalide groups, bilobalide (0.1‐10 μmol/L; Sigma‐Aldrich, St Louis, MO, USA) was added into EGM‐2. bilobalide was dissolved in DMSO and diluted with PBS beforehand. The final concentration of DMSO was below 0.05% (v/v).

A mix of terpene lactones from G. biloba: bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J (100 μg/mL each component in acetonitrile) was obtained from Sigma-Aldrich. Serial concentrations (1.5–9 μg/ml) were prepared in DMEM 2% FBS for experiments. Acetonitrile (9% and below) does not cause viral inactivation (control). Final concentrations of acetonitrile in tested cells were ≤ 0.9% (non-toxic concentration, control).

Seven constituents of Ginkgo biloba (quercetin, kaempferol, isorhamnetin, ginkgolide A, ginkgolide B, ginkgolide C, and bilobalide) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Lawrenceville, GA). William’s E medium, penicillin, streptomycin, and dimethysulfoxide (DMSO) were purchased from Sigma-Aldrich. Purified human Topo I and II enzymes, Topo I and II assay kits, and unwinding kit were from TopoGen Inc. (Port Orange, FL). Ginkgo biloba leaf extract (Lot GBE-50-001003, a tan powdered solid) was a gift from Dr. Po Chan at the National Institute of Environmental Health Sciences (Research Triangle Park, NC). A detailed characterization and analysis of major components are described in the NTP Technical Report 5785 (link). Commercial Ginkgo biloba extract products were purchased from three American manufacturers who sell herbal extracts.