Md 2018 plus

Methanol and water, LC/MS grade, were from MercK (Darmstadt, Germany). [25,26,26,26,27,27,27-D7]-cholesterol sulfate standard (CHOS-d) and cholesterol sulfate were purchased from Sigma-Aldrich (Milan, Italy). Reference sterol sulfates were synthesized in-house by chemical derivatization of sterols (24-methylene cholesterol, desmosterol, campesterol, brassicasterol, dihydrobrassicasterol, stigmasterol, fucosterol, and β-sitosterol) that were purchased from Sigma-Aldrich (Milan, Italy). HPLC for synthetic StS standard purification were performed on a JASCO system (PU-2089 Plus quaternary gradient pump equipped with a MD-2018 Plus photodiode array detector and Sedex 85 high-sensitivity LT-ELS detector). UPLC–MS analysis was performed on Q-Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with an Infinity 1290 UHPLC System (Agilent Technologies, Santa Clara, CA, USA).

The caffeine content of the extracts was analyzed using an HPLC-DAD system (Jasco, Tokyo, Japan). This system consisted of an LC-NetII/ADC hardware interface, an automatic sampler (Jasco AS-2057 Plus), a pump (Jasco PU-2089 Plus), a multi-wavelength diode array detector (DAD, Jasco MD-2018 Plus), and a column oven (Jasco CO-2060 Plus). The gradient elution used was the following: 0 min, 5% B; 40 min, 25% B; 55 min, 45% B; 60 min, 60% B; 65 min, 5% B (solvent A: 0.5% acetic acid; solvent B: 100% methanol), with a flow rate of 1.1 mL/min. The chromatographic column was a Zorbax-SB-C18 (5 μm, 250 mm × 4.6; Agilent Technologies, Santa Clara, CA, USA), at 28 °C. The DAD recorded data from 200 to 600 nm were monitored at 274 nm. For HPLC analyses, the lyophilized extracts were dissolved in H₂O (10 mg/mL) and the injected volume was 20 µL. Caffeine was used as the standard for HPLC analyses validation. The calibration curve (y = 36,096x − 227,800; R² = 0.9996) was constructed in the linear range of 1.5–800 µg/mL. The detection limit of the method was 1.24 µg/mL.

All catalysis experiments were carried out under a protective argon atmosphere with overpressure. Before the experiments, an aqueous stock solution of p-nitrophenol (0.001 M) was flushed with argon for 30 min at 25 °C in an ultrasound bath to expel any dissolved oxygen. A volume of this p-nitrophenol solution (5 mL, 0.005 mmol) was added to a vial containing the catalytic particles (~5 mg) and NaBH₄ (1.5 mmol, final concentration of 0.3 M). The reaction mixture was then stirred at 700 rpm at 25 °C in a thermoshaker (MHL23, Hettich Benelux). To follow the reaction progress, aliquots of 100 µL were taken after 0, 10, 30, 60, 120 and 180 min. The aliquots were diluted with 1 mL of distilled water and placed immediately in a freezer at −18 °C to quench any ongoing reactions. Sampled aliquots were analyzed via high-performance liquid chromatography (HPLC) with photodiode-array UV-vis detection (Jasco MD-2018 Plus, Tokyo, Japan). A reverse-phase phenyl-hexyl column (100 × 4.6 mm, 2.6 µm particle size) was used, with a 30/70 mixture of acetonitrile/HCl_aq (0.05 mmol/L) as an eluent. Chromatograms showed two main peaks. The compounds were identified via comparison with such pure standards as 4-nitrophenol (adsorption maximum at 317 nm, retention time R_t = 3 min) and 4-aminophenol (absorption maximum at 273 nm, retention time R_t = 1 min).

Optical rotations were measured on a Jasco P2000 digital polarimeter (Jasco, Cremella, Italy). UV spectra were acquired on a Jasco V-650 spectrophotometer, NMR spectra were recorded on a Bruker Avance DRX 600 (Bruker, Milan, Italy) equipped with a cryoprobe operating at 600 MHz for protons. Chemical shift values are reported in ppm (δ) and referenced to internal signals of residual protons (CD₃OD ¹H δ3.34, ¹³C 49.0 ppm). High-resolution mass spectra were acquired on a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Milan, Italy); HPLC analyses have been performed on a Jasco system (PU-2089 Plus-quaternary gradient pump equipped with a Jasco MD-2018 Plus photodiode array detector). [1-¹³C]-acetate, [2-¹³C]-acetate and [1,2-¹³C₂]-acetate, [1-¹³C]-glycolate (all sodium salts), and salicylhydroxamic acid (SHAM) were obtained from Sigma Aldrich (Milan, Italy). Solvents were purchased from VWR (Milan, Italy) and were HPLC-grade.

HT analysis was carried out with 1 mL of each extract (Section 3.6) in an HPLC-DAD-FLD system (Jasco, Tokyo, Japan), consisting of a LC-NetII/ADC hardware interface, a pump (Jasco PU-2089), an automatic sampler (Jasco AS-2057 Plus), a multiwavelength diode array detector (Jasco MD-2018 Plus) coupled to a fluorescence detector (Jasco FP-2020 Plus) and a column thermostat (Jasco CO-2060 Plus). HT was evaluated by fluorescence and monitored at λ excitation and λ emission of 280 and 330 nm, respectively. A gradient elution program using as solvents acetic acid (A, 1%) and methanol (B, 100%) was employed: 0 min, 5% B; 30 min, 25% B; 50 min, 75% B; 55 min, 100% B; 60 min, 100% B; 63 min, 5% B. A Zorbax-SB-C18 (250 × 4.6 mm, 5 μm, Agilent Technologies, Amstelveen, The Netherlands) chromatographic column was used, at 20 °C, with a flow rate of 1 mL/min and an injection volume of 20 µL. A HT calibration curve was obtained (y = 10147x + 3486.5, R² = 0.9998, 0.25–200 μg/mL). Results are presented in g/100 g of sample in fw and dw.

Polyphenols analysis was performed by LC-4000 Series Integrated HPLC Systems (JASCO, Tokyo, Japan) consisting of a column oven (model CO-2060 plus), set at 30 °C, a UV/Vis photodiode array detector (model MD-2018 plus), an intelligent fluorescence detector (model PF-2020 plus), a liquid chromatography pump (model PU-2089 plus), an autosampler (AS-2059plus) and a ChromNAV software program (JASCO, Japan). A C18 Luna column 5 μm particle size, 25 cm × 3.00 mm I.D. (Phenomenex, Torrance, CA, USA) was used, with a guard cartridge of the same material. All solvents were filtered through a 0.45 μm filter disk (Millipore Co., Bedford, MA, USA For chestnut shell HPLC mobile phase were: water:formic acid (99.80:0.20, v/v) (A) and methanol (B). Running conditions were: 0 min 95% A and 5% B; 0–45 min 55% B. The system was equilibrated between runs for 10 min using the starting mobile phase composition. The temperature was maintained at 30 °C. Each sample was analyzed three times. The flow-rate was 0.8 mL/min. The injection volume was 20 μL.