Scoparone

The bioactive compounds in AGE were analyzed using an Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent 1260 TCC (G1316A) UV-vis detector (Agilent Technologies). The mobile phase consisted of acetonitrile (solvent A) and water containing 0.1% formic acid (solvent B), and the HPLC gradient profile was as follows: 14% A at 0–15 min, 14–26% A at 15–18 min, 26% A at 18–40 min, 26–90% at 40–50 min, and 90–14% A at 50–60 min. Five microliters of AGE (30 mg/mL) was injected into a Sunfire C18 column (5 μm, 4.6 × 250 mm; Waters Corp., Milford, MA, USA) at a column temperature of 30 °C. The flow rate was 0.6 mL/min and eluted compounds were detected at 360 nm. As reference compounds used in this study, scopolin and scoparone were obtained from PhytoLab (Vestenvergsgreuth, Germany); hyperoside was obtained from HWI Analytic GmbH (Ruelzheim, Germany); scoplectin, 3,4-di-O-caffeolyquinic acid, 3,5-di-O-caffeolyquinic acid, and 4,5-dicaffeolyquinic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade solvents were obtained from Thermo Fisher Scientific.

Neocuproine, Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium alpha medium (α-MEM), fetal bovine serum (FBS), 2,5-diphenyltetrazolium (MTT), Triton X-100, RANKL, sodium tartrate, p-nitro-phenylphosphate (PNPP), fluoresceinamine-labeled chondroitin sulfate (FACS), phosphate-buffered saline (PBS, pH 7.4), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), phenylmethanesulfonyl fluoride (PMSF), dimethylsulfoxide (DMSO), chlorogenic acid, caffeic acid, isoquercitrin, and isochlorogenic acid A were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hyeroside was purchased from Hwz Analytic GmbH (Ruelzheim, Germany). Scoparone was obtained from Phytolab GmbH&Co (Vestenbergreuth, Germany). Antibodies and protein A-agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), including anti-TRAF6 (sc-7221), V-ATPase (sc-20946), and β-actin (sc-130656). A bone resorption assay kit was purchased from CosMo Bio Co., Ltd. (Tokyo, Japan). MC3T3-E1 subclone 4 and RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).