Rutin trihydrate

In this paper, 96% (v/v) ethanol (Zorka Pharma, Sabac, Republic of Serbia), n-hexane, n-heptane, acetone, ethyl acetate, chloroform, toluene, cyclohexane, 2,2-diphenyl-1-picrylhydrazyl (DPPH), methanol (HPLC grade) (Sigma Chemical, St. Louis, MO, USA), methanol, sodium carbonate (Zorka Pharma, Sabac, Republic of Serbia), Folin-Ciocalteu reagent, gallic acid (97%) (Merck, Darmstadt, Germany), rutin trihydrate (97%) (Alpha Aesar, Kandel, Germany), vanillic acid, catechin hydrate, syringic acid, epicatechin, chlorogenic acid, caffeic acid (Sigma Chemical, St. Louis, MO, USA), and butylated hydroxytoluene (BHT) (Centrohem, Stara Pazova, Republic of Serbia) were used.

Saos2 cells [30 ] were purchased from Cedarlane (Supplier ATCC, Burlington, ON, Canada). HAM-F12, 10× Trypsin, PBS, fetal bovine serum (FBS), antibiotic-antimycotic and flasks were purchased from Lonza (Mississauga, ON, Canada). Rutin trihydrate (≥97%) and 3-(4,5-dimethy-2-lthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide were purchased from Alfa Aesar (Reston, VA, USA) and hyperoside (≥98%) from Aktin Chemicals (Sichuan, China). Human Milliplex bone magnetic bead panels for bone metabolism were purchased from EMD Millipore (Billerica, MA, USA) and read by xPONENT software on Magpix Luminex (Austin, TX, USA). Optical density (OD) measurements were analyzed using a BIO-TEK Synergy HT Multi-Detection Microplate Reader (Winooski, VT, USA). Other chemicals were purchased from Sigma-Aldrich (Oakville, ON, USA).
Cells were maintained and differentiated as previously described [31 (link)]. Following one week of differentiation in plates, differentiation cell media was supplemented with 0.01 M β-glycerophosphate (day 8) until day 21 (Media 3) in addition to either vehicle (0.1% DMSO) or flavonoid dissolved in DMSO to 0.1%.
For all experiments, a DMSO control was compared to flavonoid treated cells. All experiments were normalized to cell count using Trypan Blue staining.

TB grains were provided by Bongpyung-nongwon Co. Ltd. (Gangwon-do, Korea). Ethanol, tannic acid, acetic acid, l-ascorbic acid, sodium chloride, sodium acetate, acetonitrile, mEthanol, and sodium phosphate were acquired from Samchun Pure Chemical Co., Ltd. (Pyeongtaek, Korea). Rutin trihydrate was provided by Alfa Aesar (Ward Hill, MA, USA). Sulfatase from Helix pomatia, β-glucuronidase from bovine liver, quercetin, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), mannitol, and sucrose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was provided by Welgene Inc. (Gyeongsan, Korea). Bio-Rad protein assay dye reagent from Bio-Rad Laboratories, Inc. (Hercules, CA, USA), superoxide dismutase (SOD) assay kit from Cayman Chemical Company (Ann Arbor, MI, USA), (+)-catechin, catalase (CAT) assay kit, and glutathione reductase (GR) assay kit from ENZO Life Sciences (Farmingdale, NY, USA), lipid peroxidation (malondialdehyde; MDA) assay kit from abcam (Cambridge, UK), 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) from Thermo Fisher Scientific, Inc. (Waltham, MA, USA), and l-cysteine hydrochloride monohydrate from Junsei Chemical Co., Ltd. (Kyoto, Japan) were used.