Isoquercitrin

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).

HPLC-DAD analysis was conducted using the chromatograph LaChrom Elite (Hitachi, Tokyo, Japan) equipped with an L2455 DAD detector adjusted in the range of 230 to 400 nm, L2200 injector, L2130 pump, L2300 column oven, and a C18 column (150 × 4.6 mm, 5 µm, Merck, Darmstadt, Germany). Data were obtained with EZChrom Elite software, version 3.3.2 SP1 (Santa Clara, CA, USA). Analysis conditions were according to [20 (link)]. The mobile phase consisted of a gradient of 1% phosphoric acid and acetonitrile (Table 1) with a flow rate of 0.5 mL per minute.
Samples were obtained by diluting the extract with methanol to the concentration of 4 mg/mL followed by filtration with a 0.45 μm filter (Millex, Merck KGaA, Darmstadt, Germany). Commercial standards were used in an attempt to identify the compounds present in HEMNL by comparison of retention times and ultraviolet spectra. The standards were caffeic acid, chlorogenic acid, ferulic acid, kaempferol, gallic acid, rosmarinic acid, ellagic acid, isoquercitrin, hesperetin, quercetin, resveratrol, vitexin, isovitexin, coumarin, and myricetin acquired from Sigma-Aldrich^®, hyperoside acquired from Hwi Analytik Gmbh^® (Rülzheim, Germany), and rutin from Chromadex^® (Los Angeles, CA, USA).

All reagents used were of at least analytical quality. The standards scopoletin, (+)-catechin, quercetin, kaempferol, taxifolin, eriodictyol, astragalin, isoquercitrin and esculin, as well as methanol, were procured from Merck/Sigma Aldrich (Rahway, NJ, USA). LC-MS grade acetonitrile, water and formic acid were purchased from Fisher Scientific (Geel, Belgium). Vendors for the components of the woody plant medium (WPM), hormones and vitamins are listed in the Supplementary Materials.

Ampelopsin (AMP, #42866), isoquercitrin (ISO, #17793) and rutin (RUT, #R5143 were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) at 30 mg/mL and stored at −70 °C. Calcium hydroxide was dissolved in water at 1 mg/mL and stored at 4 °C. All compounds were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell culture medium was used as a negative control in this study. All experiments were performed in triplicate in three independent experiments (n = 9).

Standards and sample solutions were using Hitachi Lachrom Elite^® HPLC system (Hitachi High-Technologies Corporation, Tokyo, Japan) equipped with a Zorbax Eclipse C18 analytical column with acetonitrile in solvent (A) and distilled water in solvent (B). The retention time of rutin (Sigma-Aldrich, St. Louis, MO, USA), isoquercitrin (Sigma-Aldrich, St. Louis, MO, USA), astragalin (Sigma-Aldrich, St. Louis, MO, USA), mulberroside A (Sigma-Aldrich, St. Louis, MO, USA), and cis-mulberroside A (Sigma-Aldrich, St. Louis, MO, USA) were compared with those in MF and MR and their mixtures (MF/MR). Acetonitrile was used in solvent A and distilled water was used in solvent B. HPLC conditions used for the analysis of standards are shown in Supplementary Table S1. All the standards used in the study were obtained from Sigma Aldrich, St. Louis, MO, USA.