Glycitin

The reference standards of daidzin, glycitin, genistin, daidzein, glycitein and genistein were all purchased from Sigma−Aldrich (St. Louis, MO, USA)
Liquid chromatography (LC)/MS−grade acetonitrile, formic acid, methanol, and water were purchased from Merck Co. (Darmstadt, Germany).
Soybean, Artemisiae annuae herba and Mori folium used in the fermentation were purchased from YiFeng TCM shop (Nanjing, China) and authenticated by Associate Professor Jianwei Chen (Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China).

Approximately 0.5 g of dry soybean seeds was ground, mixed with 10 mL of 80% methanol in distilled water followed by sonication for 20 min, and subsequently incubated at 80°C for 14 hours. The mixture was filtered through a 0.45-μm filter and transferred to 5-mL HPLC vials. Aliquots of this filtrate (20 μL) were utilized for HPLC analysis.
For HPLC, a Phenomenex C18 column (Shimadzu LC-20A, Japan, 250 mm × 4.6 mm, 5.0 μm) and binary gradient elution were used with solvent A (methanol, chromatography purity) and solvent B (distilled water). The ratio of solvent A was 15%–45% at 0–20 min, 45%–60% at 20–30 min, 60%–80% at 30–35 min, 80%–90% at 35–40 min, and 90%–15% at 40–45 min at a flow rate of 1 mL·min⁻¹. The temperature of the column was maintained at 40°C and the detector wavelength was set at 254 nm. Isoflavones were identified by comparison with authentic standards of daidzein, genistein, daidzin, genistin and glycitin (Sigma, St. Louis, MO, USA), and quantification of the isoflavones was carried out by reference to an external standard.

The LB broth and agar media were purchased from Difco (Becton Dickinson Co., Spark, MD, USA). In order to measure the antioxidants and the enzyme activities, the reagents 2,2-diphenyl-1-picrydrazyl (DPPH), 2,4,6-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,4,5-tri(2-pyridyl)-1,3,5-triazine (TPTZ) were purchased from Sigma-Aldrich Co. (St, Louis, MO, USA). Chemicals for measuring TP and TF contents (such as the Folin-Ciocalteu’s reagent and diethylene glycol) were also purchased from Sigma-Aldrich. Amongst the twelve isoflavone standards, daidzin, glycitin, genistin, daidzein, glycitein, genistein, malonyldaidzin, malonylglycitin, malonylgenistin, acetyldadzin, acetylglycitin, and acetyldaidzin were purchased from Sigma-Aldrich and the LC Laboratories (Woburn, MA, USA). For the analysis, the reagents and solvents (such as HPLC-grade water, methanol, acetonitrile, glacial acetic acid, etc.) were purchased from Sigma-Aldrich and Fisher Scientific International, Inc. (Fairlawn, NJ, USA), respectively.