Mak187

The scavenging activity of 2,2-diphenyl-1-picrylhydrazine (DPPH) was analyzed using spectrophotometric methods [25 (link),26 (link)] with minor modifications. This study utilized the stable free radical DPPH (Sigma-Aldrich, St. Louis, MI, USA, Pcode: 101845869). In a 1.5-mL tube, 50 μL of each plasma and milk sample was mixed with 1 mL of DPPH reagent methanol solution (25 μmol/L). The mixture was thoroughly shaken and incubated in the dark at room temperature for 30 min, followed by centrifugation at 4000 r/min at 4 °C for 10 min. We then transferred the supernatant to a 96-well plate with 200 μL per well, and measured the absorbance at 517 nm using a microplate reader (Epoch, BioTek, Luzern, Switzerland). The DPPH-scavenging activity was calculated as a percentage using the following formula:
where Ac represents the control absorbance, and As represents the sample absorbance.
The enzyme activity of the total antioxidant capability (TAC), superoxide dismutase (SOD), glutathione S-transferase (GST), and malondialdehyde (MDA) was measured using Sigma-Aldrich kits, namely, MAK187 for TAC, 19160 for the SOD determination, CS0410 for GST, and MAK085 for MDA. Subsequently, the enzyme activity of all samples was determined using a microplate reader (Thermo Scientific™, Waltham, MA, USA).

For the third set of experiments, PBMCs were again centrifuged at 300× g for five minutes and were suspended in PBS supplemented by protease inhibitors to be sonicated twice for 10 s in ice, with a break of 30 s. The activity of H6PD and G6PD were assayed using an absorbance microplate reader (ELx808™, Winooski, VT, USA) to follow the reduction of NADP at 340 nm [24 (link),25 (link),26 (link),33 (link)]. H6PD enzymatic function was tested in the presence of Tris-HCl pH 7.4 100 mM, glucose 10 mM, and NADP 0.5 mM. By contrast, G6PD activity was assayed in the presence of Tris-HCl pH 7.4 100 mM, glucose-6-phosphate (G6P) 10 mM, and NADP 0.5 mM.
Malondialdehyde (MDA) levels were evaluated, by the thio-barbituric acid reactive substances assay, using a UV/visible spectrophotometer (Ultraspec 2000, Pharmacia Biotech, Erie, PA, USA) [26 (link),34 (link)]. In all cases, enzymatic activity was normalized for total protein concentrations tested using Bradford analysis [35 (link)].
Finally, total antioxidant capacity was evaluated following the manufacturer’s instructions of a dedicated kit (MAK187, Sigma, St. Louis, MO, USA) that provides a complete description of the total cell antioxidant power associated with the endogenous scavengers, expressed as Trolox equivalent antioxidant capacity content.

A series of biomarkers were used to assess the potential toxic effects of REEs on Phytolacca species. First, leaf pigments (chlorophylls, flavonoids and anthocyanins) and the nitrogen balance index (NBI) were quantified using a DUALEX^® SCIENTIFIC⁺ apparatus^{58 (link)} (Force-A, Orsay, France) before harvesting. Forty technical replicates were carried out per plant. Second, root architecture was analysed. Root systems were photographed at harvest. The analyses of lateral root length and lateral root density (number of lateral roots per cm of primary root) were carried out using the RootNav software^{59 (link)}. Finally, the leaf and root contents of malondialdehyde (MDA) and total antioxidant compounds (TAC) were assayed from 50 mg of frozen powder using commercial kits (MAK085 and MAK187, Sigma-Aldrich, France) according to the manufacturer’s protocols. Two technical replicates were performed per sample.