Morroniside

Testosterone propionate (TP) was purchased from Wako Pure Chemicals (Tokyo, Japan). Finasteride was supplied from Merck & Co., Inc. (Kenilworth, NJ, USA). Antibodies against androgen receptor (AR, sc-816), proliferating cell nuclear antigen (PCNA, sc-56), E2F1 (sc-193), Rb (sc-377528), cyclin D1 (sc-753), and β-actin (sc-81178) were obtained from Santa Cruz Biotechnology, Inc. (TX, USA). Antibody against prostate-specific antigen (PSA, Catalog number, PB9259) was purchased from Boster Biological Technology (CA, USA). Loganin (≥97.0%), psoralen (≥99.0%), and isopsoralen (≥95.0%) were procured from Sigma-Aldrich (Seoul, Korea). Morroniside (≥97.0%) was purchased from ChemFaces (Wuhan, Hubei, China). HPLC-grade acetonitrile and water were procured from JT&Baker (Seoul, Korea).

Reverse-phase UHPLC was performed on a Waters ACQUITYUHPLC H-Class system (Milford, MA, United States) consisting of a quaternary solvent manager pump, sample manager – TN and photodiode array (PDA) detector. Empower 3 Pro software (Milford, MA, United States) was used for UHPLC data analysis. Chromatographic separation was accomplished on a Perkin Elmer C18 reverse-phase column (Brownlee SPP 100 mm × 1.0 mm I.D., 1.7 μm) at 30°C and monitored at 283 nm for 5-HMF (5-hydroxymethyl-2-furaldehyde, Sigma Aldrich), 231 nm for paeoniflorin (MFDS, Osong, South Korea), 240 nm for morroniside (ChemFaces, Wuhan, China), and 237 nm for loganin (MFDS). A solvent system consisting of acetonitrile (solvent A) and 0.1% formic acid DW (solvent B) was used with a gradient from 2% (solvent A): 98% (solvent B) to 100% (solvent A): 0% (solvent B) over 18 min at a flow rate of 0.4 ml/min. Standard stock solutions were prepared by dissolving standards in 100% methanol to obtain a final concentration of 10 μg/ml. After ultracentrifugation at 9000 rpm and filtration through a syringe filter (0.2 μg, Whatman^TM, Maidstone, United Kingdom), 4 ml of sample was injected six times.