Arium 611 di

Deuterated methanol (purity 99.8%) and deuterium oxide (99.9%) were supplied from Deutero GmbH (Kastellaun, Germany). Trimethyl silylpropionic acid sodium salt-d₄ (TSPA-d₄), acetonitrile, acetic, formic acids, methanol, and DMSO of the HPLC grade and CDDO-Me were purchased from Merck KGaA (Darmstadt, Germany). Deionized water was purified through Ultrapure Water Systems Arium^® 611DI (Sartorius AG, Gottingen, Germany). Myconoside and calceolarioside E were isolated and purified within the current study. The CDDO-Me and myconoside were dissolved in DMSO, while calceolarioside E was dissolved in methanol. The compounds were then diluted with PBS (pH 7.4) to a concentration of 1 mg/mL, passed through a sterile 20 µm syringe filter (Corning GmbH, Kaiserslautern, Germany), and stored at −20 °C.
Column chromatography (CC) on Polyamide 6 (Fluka, Germany), Sephadex LH-20 and Lobar chromatography (Lobar RP-18, Merck) were used for separation and purification of the individual compounds. Preparative thin layer chromatography (PTLC) was performed on pre-coated plates 60 F254, 0.25 mm and thin layer chromatography (TLC) was performed on 60 F254 plates (Merck). Separation was visualized by spraying with 20% (v/v) H₂SO₄ in an ethanol solution.

Approximately 0.2 g of each of the dried soil sample was digested for 20 min at 140 °C and 15 atm in 5 mL of concentrated nitric acid (‘suprapur’ grade), 2 mL hydrochloric acid, 1 mL hydrofluoric acid, and 1 mL of 30% w/v hydrogen peroxide. The digested samples were cooled to room temperature and transferred to a Teflon cup, 1 mL perchloric acid was added, and the hydrofluoric acid was removed for 10 min at 180 °C, and then used for the analysis of total selenium in the soil [10 (link)].
Approximately 0.2 g of each of the dried herbage samples was digested for 5 min at 140 °C and 15 atm in 5 mL of concentrated nitric acid (‘suprapur’ grade) and 1 mL of 30% w/v hydrogen peroxide. A total of 200 μL of each serum sample was digested for 4 min at 140 °C, at 14 atm in 5 mL of concentrated nitric acid (‘suprapur’ grade) in a microwave digestion system (WX-4000, Shanghai Qiyao Ltd. Co). The digested samples were transferred to polypropylene sample tubes and diluted to 100 mL with ‘ultrapure’ water (Sartorius Arium 611 DI) [10 (link)].
Samples (soil, herbage, and serum) were analyzed for Ca, Mg, K, Na, P, Fe, Zn, Mn, and Cu by the ICP-AES analyzer (IRIS Advantage ER/S) [16 (link)], and the analysis of Se was carried out by atomic fluorescence spectrophotometry [17 ].