Methanol

Dimethyl sulfoxide (DMSO), methanol, and ethanol were obtained from Carlo Erba Reagent (Milan, Italy) in their highest commercially available purity grade. DMT (code #31298-250MG), TPA (code #40818-100MG), and all other reagents, if not differently specified, were purchased from Merk Life Science (Milan, Italy).

Folin-Ciocalteu reagent, sodium carbonate, sodium nitrite, aluminum chloride, DPPH, caffeic acid, caffeoyquinic acid, p-coumaric acid, protocatechuic acid, kaempferol, hyperoside, rutin hydrate, and dimethylsufoxide (DMSO) were purchased from Sigma Aldrich (St. Louis, MO, USA). Solvents such as acetone, acetonitrile, methanol. and water (HPLC-MS grade) were purchased from Carlo erba (Peypin, France).

For sample preparation, 5 g of walnuts were homogenized in an Ultraturrax T-18 basic for 2 min, at 24,000 rpm with 20 mL of a 3% methanol (Carlo Erba Reagents, France) + formic acid (Normapur, NWR) solution. The extracts were centrifuged for 10 min at 4000 x g in a Heraeus Biofuge Stratos centrifuge. The supernatant, methanolic extract, was separated from the precipitate and filtered through Whatman # 4 filter paper and collected in opaque flasks. The precipitate was subjected to a new extraction process under identical conditions, and its methanolic extract was joined to that obtained after the first extraction.
The content of total phenolic compounds (TPC) of walnut extracts was determined using the Folin–Ciocalteu method [31 ]. The reaction mixture contained 1 mL of walnut extracts, 5 mL of the Folin–Ciocalteu reagent (Merck; Darmstadt, Germany, and 20 mL of sodium carbonate (200 mg/L) (Scharlau, Sentmenat, Spain). The final volume was made up to 50 mL with distilled water. After 30 min of reaction, the absorbance at 765 nm was measured in a spectrophotometer Varian Cary 50 Bio (Varian, Palo Alto, CA, USA). The determinations were carried out in triplicate, and TPC content was expressed as g of gallic acid/L by using a calibration curve of pure gallic acid (Sigma-Aldrich, St. Louis, MO, USA).

Glyphosate, aminomethylphosphonic acid, and malonic acid were purchased from Merck (Darmstadt, Germany). Ammonia solution (30%) and methanol were purchased from Carlo Erba (Milan, Italy). Standard solutions of phosphorous (10,000 mg L⁻¹) were obtained from Inorganic Ventures (Lakewood, NJ, USA).
HPLC-grade water was obtained from an ultrapure water purification system (18.2 MΩ cm, ELGA PURELAB flex, Veolia Water Solutions and Technologies, Ontario, Canada).

The peptides PSELT (H-Phe-Gln-Ile-Cys-Val-Ser-Sec-Gly-Tyr-Arg-OH), [Ser46,49] PSELT [called inert PSELT (I-PSELT) as a control], and PSELT-dansyl were synthesized by Fmoc solid phase methodology on an automated peptide synthesizer (CEM, Saclay, France) as previously described [19 (link)]. KCl, NaCl, NaHCO₃, CaCl₂, MgSO₄, KH₂PO₄, NaH₂PO₄, Na₂HPO₄, mannitol, glucose, Na-pyruvate, β-nicotinamide adenine dinucleotide (NADH), reduced disodium salt hydrate, 2,4 dinitrophenylhydrazine (DNPH), 2-thiobarbituric acid (TBA), bovine serum albumin (BSA), butylated hydroxyanisole, diethyl ether, ethylenediaminetetraacetic acid (disodium salt), diethylenetriamine pentaacetic acid, pyrogallol, streptomycin sulfate, tween-20, hydrogen peroxide (H₂O₂), and urea were purchased from Sigma Aldrich (St. Louis, MO, USA). Tunicamycin (TM) was from Cayman Chemical (Ann Arbor, MI, USA), Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12), penicillin/streptomycin and fetal bovine serum (FBS) were provided by Thermo Fisher Scientific (Waltham, MA, USA). All the solutions were freshly prepared before starting the experiments. Absolute ethanol, ethyl acetate, hydrochloric acid, methanol, and trichloroacetic acid (TCA) were from Carlo Erba Reagents (Cornaredo, Milano, Italy).