Daidzein

The standards for anthocyanins comprised delphinidin 3-O-glucoside chloride, petunidin 3-O-glucoside chloride, cyanidin chloride (Extrasynthese, Genay, France), and cyanidin 3-O-glucoside chloride (SinoStandards Bio-Tech Co., Ltd., Chengdu, China). The standards for flavan-3-ols consisted of catechin hydrate (Sigma Aldrich Co., St Louis, MO, USA) and epicatechin (Chemfaces, Wuhan, China). The standards for isoflavones comprised daidzein, glycitein, genistein, daidzin, glycitin, genistin (LC Laboratories, Woburn, MA, USA), acetyl daidzin, acetyl glycitin, acetyl genistin (Nacalai Tesque, Kyoto, Japan), malonyl daidzin, malonyl glycitin, and malonyl genistin (GenDEPOT, Katy, TX, USA).

daidzein, genistein, and their derived metabolites dihydrodaidzein, dihydrogenistein, O-DMA, and equol were detected and quantified by UHPLC based on the method for isoflavone determination in urine samples reported by Redruello et al. [37 (link)]. Briefly, the control strains were independently cultured in GAM-Arg medium supplemented with 12.5–100 µM daidzein or genistein (LC Laboratories, Woburn, MA, USA). Furthermore, the selected strains were inoculated in pairs, triads, and tetrads and cultured with 100 µM of each isoflavone as above. After overnight incubation, cultures were centrifuged at 16,000× g for 2 min, and then filtered through a 0.2 µm polytetrafluoroethylene (PTFE) membrane (VWR, Radnor, PA, USA). The culture supernatants were used directly in UHPLC analyses. Quantification was performed against calibration curves for isoflavone and isoflavone-derived standards obtained from a commercial source (LC Laboratories). In this work, the limit of quantification (LoQ) for the different compounds analyzed were, in µM, 6.25 for daidzein, genistein, and dihydrogenistein, 5.62 for O-DMA, 3.13 for dihydrodaidzein, and 3.12 for equol.

Cocaine hydrochloride was obtained from the Spanish Agency of Medicines and Medical Devices (AEMPS, Madrid, Spain), dissolved in sterile saline, and administered at 0.5 mg/kg (intravenously, i.v.). All natural isoflavones (daidzin, daidzein, and genistein) were purchased from LC Laboratories (Woburn, MA) or PhytoLab (Vestenbergsgreuth, Germany), dissolved in 50 μl of Tween 80/0.5% carboxymethylcellulose (CMC) in sterile saline and administered at different doses by intraperitoneal (i.p.) route. Disulfiram was purchased from TOCRIS (Bio-Techne, Minneapolis, MN), dissolved in 50 μl of Tween 80/0.5% CMC in sterile saline and administered at different doses by i.p. route. Phenylephrine (1 mg/kg, subcutaneous (s.c.), not permeable to cross the Blood Brain Barrier), atipamezole (0.4 mg/kg, s.c.), isoproterenol (0.25 mg/kg, i.p.), SKF38393 (0.1 mg/kg, s.c.), quinpirole (0.01 mg/kg, i.p.) and tamoxifen (1 mg/kg, i.p.) were purchased from TOCRIS and dissolved in 50 μl of Tween 80/0.5% CMC in sterile saline.

The HPLC-grade acetonitrile and water (Daejung Chemical & Metals Co., Siheung, Korea) were used as mobile phases for isoflavones analysis. Standards of isoflavone aglycones (daidzein, glycitein, and genistein) and β-glycosides (daidzin, glycitin, and genistin) were purchased from LC Laboratories (Woburn, MA, USA). Isoflavone acetylglycosides (acetyldaidzin, acetylglycitin, and acetylgenistin) were obtained from Nacalai tesque (Kyoto, Japan), and malonylglycosides (malonyldaidzin, malonylglycitin, and malonylgenistin) were obtained from GenDEPOT (Katy, TX, USA).