Ascorbic acid aa

The antioxidant capacity was investigated by a DPPH^• radical scavenging in vitro assay [41 (link)]. The activity of the standard or EE was determined by adding 100 μL of ethanol 95% (v/v) (Vetec, Recife, Brazil), 100 μL of sodium acetate buffer 100 mM (pH 5.5, Merck,, Darmstadt, Germany), 50 μL of 507.2 μM DPPH (Sigma-Aldrich, Jurubatuba, Brazil) radical solution in ethanol, and 10 μL of standard or sample to 96-well plate. After 15 min, the spectrophotometer was read, discounting the blank. For the blank, 150 μL of ethanol 95% (v/v), 100 μL of sodium acetate buffer (pH 5.5), and 10 μL of standard or sample were added in the same concentration. The absorbance was determined at 517 nm using a Multimode Plate Reader (EnSpire, Perkin-Elmer, Singapore). Ascorbic acid (AA, Merck, Darmstadt, Germany) was used as positive control (0.6–6 µg/mL). The results were expressed as the efficient concentration that can scavenge DPPH^• radical at 50% (EC₅₀). All analyses were performed in triplicate, and data were expressed as mean ± standard deviation.

Human NPCs were derived from human fibroblast iPSC by treatment with small molecules as described before [14 (link),15 (link)]. Human induced pluripotent stem cell derived neural progenitor cells used in this study were generated at the Max Planck Institute for Molecular Biomedicine (Münster, Germany) [15 (link)]. NPCs were cultured on Matrigel-coated 12-well cell-culture plates (Nunc, Rochester, New York, NY, USA) in N2B27 medium supplemented with 3 µM CHIR99021 (Axon Medchem, Groningen, The Netherlands), 0.5 µM Smoothened agonist (SAG) (Cayman Chemical, Ann Arbor, MI, USA), and 150 µM Ascorbic Acid (AA; Merck, Darmstadt, Germany) with a medium change every second day. For plate coating, Matrigel (high concentration, growth factor reduced; Corning, New York, NY, USA) was diluted 1:100 in Knockout Dulbecco modified eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) for coating with 500 µL per well for 2 h. Cells were split in a 1:9 to 1:12 ratio every 7 days by single cell digestion with prewarmed accutase (Merck) for 10 min at 37 °C. Cells were diluted 1:10 in DMEM (Merck) prior to centrifugation at 200× g for 5 min. After resuspension in fresh NPC medium (N2B27 supplemented with CHIR, SAG, and AA), cells were plated on Matrigel-coated cell-culture dishes.

Analytical grade silver nitrate (AgNO₃, 99.8%, Merck, KGaA, Darmstadt, Germany), D-(+) raffinose pentahydrate (Alfa Aesar, Karlsruhe, Germany), and sodium hydroxide (NaOH, 99%, Merck, Germany) were used to prepare aqueous dispersion of silver nanoparticles. The stock standard solutions of 500 mg L⁻¹ Cr(VI) and 500 mg L⁻¹ Cr(III) were prepared using K₂Cr₂O₇ (Sigma-Aldrich, GmbH, Sternheim, Germany) and CrCl₃·6H₂O (Merck, Germany), respectively. Working standard solutions for Cr(VI) within the concentration range of 1 × 10⁻⁶–1 × 10⁻⁴ mol L⁻¹ were prepared daily by appropriate dilution of the stock standard solution using 1.5 × 10⁻³ mol L⁻¹ HCl. All diluted Cr(VI) solutions were kept refrigerated at 4 °C. Analytical grade ascorbic acid (AA) and salts of the different cations studied (NaCl, KCl, MgCl₂, CaCl₂, Pb(NO₃)₂, ZnCl₂, CuCl₂, NiCl₂, CdCl₂, CoCl₂, FeCl₃) were purchased from Merck, Germany. Doubly distilled water was used for preparation of all reagent solutions.